Disabled Gene Is Involved in CML Progression and Its Expression Level at Diagnosis Can Predict Major Molecular Response (MMR) to Imatinib Therapy.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3964-3964
Author(s):  
Daniela Cilloni ◽  
Francesca Arruga ◽  
Francesca Messa ◽  
Monica Pradotto ◽  
Enrico Bracco ◽  
...  
Keyword(s):  
Western Blot ◽  
Conflicts Of Interest ◽  
Fragile Site ◽  
Blast Crisis ◽  
Adaptor Protein ◽  
Molecular Response ◽  
Loss Of Function ◽  
Imatinib Resistance ◽  
Transcript Levels

Abstract Abstract 3964 Poster Board III-900 Despite the role of Bcr-Abl in the pathogenesis of Chronic Myeloid Leukemia (CML) is well established, the mechanisms leading to CML progression remain unknown. By using our model of Drosophila melanogaster (Dm) transgenic for human Bcr-Abl we have identified Dab1 and Dab2 as genes involved in CML progression. In Dm the loss of function of these genes induced a worsening of the phenotype generated by hBcr-Abl expression. Moreover, an even stronger phenotype was obtained by silencing Disabled using RNAi fly strains. By contrast, Dab gain of function rescued Bcr-Abl phenotype. Disabled is a non receptor tyrosine kinase, identified in Dm as an adaptor protein acting downstream of many tyrosine kinases receptors (RTK). One of the human homolog of Disabled, Dab1 is a large common fragile site gene, involved in neural migration and the other homolog Dab2 codes is an adaptor protein implicated in RTK signalling, endocytosis, cell adhesion and differentiation. The downregulation of both genes is described in many types of cancer suggesting their possible role in oncogenesis but their involvement in haematological malignancies has never been described. The aim of the study was to investigate the role of Dab1/2 in CML progression. Dab1 and Dab2 mRNA was analyzed by Real Time PCR in 94 samples from 82 CML patients (34 PB and 60 BM). Among those patients 55 were collected at diagnosis (19 enrolled in TOPS study), 9 patients in CP collected at the time of imatinib resistance, 7 in accelerated phase and 11 in BC, In addition 21 healthy donors (10 PB and 11 BM). In 18 patients, genes expression was analyzed during remission as well. Protein expression was investigated by Western Blot and Immunofluorescence. In addition, K562 cells were transfected with Dab plasmids to gain insight about the effects of Dab1/2 on cell proliferation and apoptosis. We found that in CML patients Dab1/2 expression levels were significantly decreased in either BM or PB (p<0.002 and p<0.0004) as compared to healthy subjects. Furthermore, in blast crisis Dab1/2 transcript levels are further decreased. In contrast, at the time of remission, the transcript levels were comparable to normal values. Data analysis of patients included in TOPS studies shows that Dab1 values are lower among those achieving MMR by 12 months (median value 2) compared to those without MMR (median 4,59) although this difference does not appear significant (p=0.15). By analyzing all together the CP patients enrolled in different protocols based on imatinib as front line therapy, this difference became significant (p=0,01). Western Blot and immunofluorescence confirmed the absence of Dab1/2 proteins in course of active CML samples while it reappeared during remission. Dab1 and Dab2 transfection of K562 significantly reduced proliferation (p=0,002). In conclusion, our results show a significant decrease of Dab1/2 expression in blast crisis samples, when compared to CP CML and healthy volunteers, which suggest a role of Dab1/2 in slowing down or suppressing a progression. Among CP CML patients the responders to TKI therapy have been detected to express a lower Dab levels of than non responders. We find those data intriguing and suggesting that molecular factors involved in the regulation of CML progression could be uncoupled from the mechanisms regulating response to TKI therapy. Supported by Novartis Oncology, Clinical Development, TOPS Clinical Correlative Studies Network Disclosures: No relevant conflicts of interest to declare.

Identification of Rab5 as a Gene Involved in Chronic Myeloid Leukemia (CML) Progression.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3470-3470
Author(s):  
Daniela Cilloni ◽  
Monica Pradotto ◽  
Francesca Messa ◽  
Francesca Arruga ◽  
Enrico Bracco ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Western Blot ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Blast Crisis ◽  
Small Gtpases ◽  
Loss Of Function ◽  
Imatinib Resistance ◽  
Protein Levels

Abstract Abstract 3470 Poster Board III-358 The role of Bcr-Abl in the pathogenesis of Chronic Myeloid Leukemia (CML) is well established, however, the mechanisms leading to CML progression remain poorly understood. By using our model of transgenic Drosophila Melanogaster (Dm) for human Bcr-Abl driven CML we have identified Rab5 as a gene involved in the regulation of CML progression. The Rab5 is a member of gene family small GTPases which are involved in the regulation of vesicular transport. Lately several important reports have linked some members of the Rab family to invesivness and migration of cancer cells. Rab5 is associate with alpha-integrin subunits and modulates their endosomal traffic and subcellular localization. We have observed that a loss of function of Rab5 gene have induced a worsening of the CML phenotype generated by hBcr-Abl expression. In contrast, Rab gain of function rescued Bcr-Abl phenotype. The aim of the study was to evaluate the expression of Rab5 in CML cells to better understand if a potential correlation with progression, which has been observed in the model, could be confirmed in patients. Methods Rab5 gene expression was measured by Real Time PCR in 90 samples from 80 CML patients (32 PB and 58 BM). Among those, 53 are collected at diagnosis (19 of 53 patients have been enrolled in TOPS study). In addition, 9 samples from in CP patients have been collected at the time of imatinib resistance, 7 in accelerated phase and 11 in BC. In 14 patients, genes expression was analyzed during remission as, well. In parallel, 21 healthy donors (10 PB and 11 BM) have been evaluated. Rab5 protein expression was investigated by Western Blot and Immunofluorescence. We have also utilized K562 transfected with Rab5 plasmid, which we have generated to gain insight about the effects of Rab5 on cell proliferation and apoptosis. Results Rab5 transfection and overexpression in K562 significantly reduced proliferation and affected apoptosis. We found that in CML patients Rab5 expression levels were significantly decreased in either BM or PB (p<0.001 and p<0.0001) as compared to healthy subjects. Furthermore, in blast crisis samples we have found Rab5 transcripts levels to be further decreased. In contrast, at the time of remission, the transcript levels were comparable to normal values. Our preliminary analysis of samples from TOPS trial have shown a trend that Rab5 levels are lower among those patients achieving MMR by 12 months, when compared to the group of patients non achieving MMR on 400 mg, but that difference was not statistically significant (p=0.2). Among those randomized to receive imatinib 800 mg the difference was statistically significant with a median value among those achieving MMR of 1.27 vs 2.14 in the group without MMR (p=0.04). The protein levels have been analyzed by Western Blot and immunofluorescence and allow us to show detectable levels of Rab5 in samples collected at remission, but undetectable levels in course of active CML disease. Although preliminary, our results show a significant decrease of Rab5 expression in blast crisis samples, when compared to CP CML and healthy volunteers, which suggest a role of Rab5 in slowing down or suppressing a progression. Surprisingly, among CP CML patients the responders to TKI therapy have been detected to express a lower level of Rab5 than non responders. We are conducting further studies to better explain these data, which we find intriguing and suggesting that molecular factors involved in the regulation of CML progression could be uncoupled from the mechanisms regulating response to TKI therapy. Supported by Novartis Oncology, Clinical Development, TOPS Clinical Correlative Studies Network Disclosures No relevant conflicts of interest to declare.

scholarly journals The Role of Monitoring the Bcr-Abl Transcript Levels in the Management of Patients with Chronic Myeloid Leukemia

2013 ◽  
Vol 59 (2) ◽  
pp. 71-74
Author(s):  
Aliz-Beáta Tunyogi ◽  
I Benedek ◽  
Judit Beáta Köpeczi ◽  
Erzsébet Benedek ◽  
Enikő Kakucs ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Kinase Inhibitors ◽  
Blast Crisis ◽  
Chronic Gvhd ◽  
Molecular Response ◽  
Imatinib Treatment ◽  
Molecular Monitoring ◽  
Hematopoietic Stem ◽  
Transcript Levels

Abstract Introduction: Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder; the molecular hallmark of the disease is the BCR-ABL gene rearrangement, which usually occurs as the result of a reciprocal translocation between chromosomes 9 and 22. Tyrosine kinase inhibitors (TKI) were the first drugs that targeted the constitutively active BCR-ABL kinase and it have become the standard frontline therapy for CML. Monitoring the treatment of CML patients with detection of bcr-abl transcript levels with real time qualitative polymerase chain reaction (RQ-PCR) is essential in evaluating the therapeutic response. Material and method: At the Clinical Hematology and BMT Unit Tîrgu Mureș, between 2008-2011, we performed the molecular monitoring of bcr-abl transcript levels with RQ-PCR in 16 patients diagnosed with CML. Results: We have 11 patients on imatinib treatment who achieved major molecular response. One patient lost the complete molecular response after 5 years of treatment. Two patients in blast crisis underwent allogeneic hematopoietic stem cell transplantation from identical sibling donors. The first patient is in complete molecular remission after 4 years of the transplant with mild chronic GVHD. The other patient had an early relapse with treatment refractory disease and died from evolution of the disease. Three patients with advanced phases of the disease present increasing transcript levels. We performed the dose escalation, and for two of them the switch to the second generation of TKI. Conclusions: Regular molecular monitoring of individual patients with CML is clearly desirable. It allows for a reassessment of the therapeutic strategy in cases of rising levels of BCR-ABL as an early indication of loss of response.

miRNA29a Regulates Oncogenic SKI Expression in Acute Myeloid Leukemia (AML).

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1968-1968
Author(s):  
Sabine Teichler ◽  
Thomas Illmer ◽  
Thorsten Stiewe ◽  
Josephine Roemhild ◽  
Andreas Neubauer
Keyword(s):  
Western Blot ◽  
Conflicts Of Interest ◽  
Fragile Site ◽  
Micro Rna ◽  
Chromosome 7 ◽  
Hl60 Cells ◽  
Rna Profiling ◽  
Micro Rnas

Abstract Abstract 1968 Poster Board I-991 Introduction: AML patients with deletion of chromosome 7 (−7) or deletion of 7q (−7q) have a poor prognosis. We have found that the nuclear oncogene SKI is overexpressed in AML, especially in AML with −7/−7q. SKI acts in AML as a repressor of retinoic acid induced myeloid differentiation (Ritter et al., (2006) Leukemia). As we found SKI up regulated in AML, we asked how SKI expression may be regulated. The aim of our study was to find a molecular background for increased SKI level. On chromosome 7 is a cluster of micro-RNAs (miRNAs) localized particularly around the fragile site 7q32 (Calin et al., (2003) PNAS). Therefore we investigated whether there exists a link between expression of miRNAs localized on chromosome 7 and up regulation of SKI expression in AML. Methods: We used micro RNA profiling analysis, FACS, Western blot, RQ-PCR and luciferase assays to determine the role of miRNA29a in regulating SKI expression. Results: We found that the expression of miRNA25, miRNA29a, miRNA183 and miRNA335 was downregulated in AML patients with -7/-7q. Transfection studies with these four miRNAs in HL60 cells revealed in FACS that miRNA29a inhibits SKI expression (60,4%) compared to nonsense control (100%) and other miRNAs (miRNA25: 91%, miRNA183: 101%, miRNA335: 93%). Western blot experiments confirmed that miRNA29a reduces SKI level in HL60 cells. In keeping, miRNA29a also represses expression of the SKI target gene Nr-CaM in IFB melanoma cells. Knock down of miRNA29a using miRNA29a inhibitor molecules induces SKI expression in the high miRNA29a and low SKI expressing cell line NW1539. Luciferase assays in NW1539 and HeLa transfected with 3′UTR-constructs and HeLa cells cotransfected with miRNA29a demonstrated that miRNA29a binds to 3′UTR of SKI in vitro. Furthermore, comparison of SKI and miRNA29a expression of AML patient samples indicates that miRNA29a expression is associated with low SKI level in vivo. Conclusion: Our data show that miRNA29a which is located on 7q32 regulates expression of the oncogene SKI in vitro and in vivo. We suggest the deletion of miRNA29a as mechanism for up regulation of SKI in AML with -7/-7q and thus propose that in AML, this effect may contribute to the tumor suppressive function of miRNA29a. Disclosures: No relevant conflicts of interest to declare.

Re-Expression of the AML1/ETO Target Gene LAT2/NTAL/LAB Results In Direct Interference with Myeloid Differentiation In AML1/ETO-Positive Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2474-2474
Author(s):  
Jesus Duque-Afonso ◽  
Aitomi Essig ◽  
Leticia M Solari ◽  
Tobias Berg ◽  
Heike L. Pahl ◽  
...  
Keyword(s):  
Western Blot ◽  
Cell Lines ◽  
Functional Role ◽  
Target Gene ◽  
Conflicts Of Interest ◽  
Myeloid Cell ◽  
Leukemia Cells ◽  
Myeloid Differentiation ◽  
Granulocytic Differentiation

Abstract Abstract 2474 Background: The leukemia-specific oncofusion protein AML1/ETO regulates different target genes, including the LAT2 gene (encoding the adaptor molecule LAT2/NTAL/LAB), which is epigenetically repressed by AML1/ETO as we have previously described. LAT2 is phosphorylated by c-kit and has a role in mast cell and B cell activation. To address the functional role of LAT2 during myeloid differentiation, expression studies were performed in myeloid cell lines, and LAT2 was overexpressed by retroviral gene transfer in AML1/ETO-positive Kasumi-1 cells and AML1/ETO-negative U-937 cells. Methods: To induce monocytic and granulocytic differentiation, the myeloid cell lines U-937, HL-60 and NB4 were treated with PMA and ATRA, respectively, and LAT2 expression measured by both Northern and Western blot. LAT2 was overexpressed in Kasumi-1 and U-937 cells by use of the retroviral vector pMYSiG-IRES-GFP. Virus was produced in 293T cells and titrated in TE671 cells. Following transduction, GFP-positive cells were sorted by fluorescence-activated cell sorting (FACS). Transduced cells were treated with PMA (2 and 10 nM for 24 and 48 hours) and ATRA (0.1 μM and 0.5 μM for 48 and 96 hours), respectively. Results: The AML1/ETO-negative myeloid cell lines HL-60, NB4 and U-937 readily expressed LAT2, which was further upregulated by PMA, and transiently downregulated with ATRA. In the AML1/ETO-positive Kasumi-1 and SKNO-1 cells, LAT2 expression was absent. To address the functional role of this repression, forced expression of LAT2 was achieved in Kasumi-1 and U-937 cells and resulted in effective processing of LAT2 protein (confirmed by Western blot), and a decrease in the expression of the differentiation markers CD11b and CD11c (FACS analysis) in Kasumi-1 but not U-937, with only minor effects of LAT2 overexpression upon apoptosis and cell growth arrest. Notably, during both PMA- and ATRA-induced differentiation, a striking maturation block occurred in Kasumi-1 (measured by CD11b/CD11c expression, observed at different doses and time points of these treatments), while differentiation of U-937 cells was unimpaired by overexpression of LAT2. Conclusions: In AML1/ETO-negative leukemia cells, LAT2 expression is differentially regulated during monocytic and granulocytic differentiation. In AML1/ETO-positive leukemia cells, in which LAT2 is repressed, LAT2 re-expression imposes a striking maturation block. Graded expression of this novel AML1/ETO target gene may therefore play an important role in maintaining the phenotypic characteristics of this leukemia subtype. Disclosures: No relevant conflicts of interest to declare.

Stimulation of the Mineralocorticoid Receptor by Aldosterone Leads to Activation of HL-60 Neutrophils,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3206-3206
Author(s):  
Carlos E Vazquez ◽  
Gregory N Prado ◽  
Enrique R Maldonado ◽  
Gabriela Saca ◽  
Iren M Ortiz ◽  
...  
Keyword(s):  
Western Blot ◽  
Respiratory Burst ◽  
Mineralocorticoid Receptor ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Neutrophil Activation ◽  
Human Neutrophils ◽  
Dead Cell ◽  
Cardiovascular Morbidity And Mortality

Abstract Abstract 3206 Blockade of the mineralocorticoid receptor (MR), the receptor for aldosterone (ALDO), improves cardiovascular morbidity and mortality. There is growing evidence for a critical role of ALDO in inflammation in addition to its well-described effects on sodium homeostasis. However, the role of ALDO on neutrophil activation is not entirely clear. We studied the role of ALDO on HL-60, a human promyelocytic cell line, induced to differentiate into neutrophil-like cells by incubation for 3 days with 1.3% DMSO. We detected the presence of the mineralocorticoid receptor (MR), the receptor for ALDO, by western blot analyses and MR transcript by quantitative RT-PCR using TaqMan detection probes in these cells. Cells incubated with ALDO (10−8-10−7 M) showed a dose-dependent rise in cytosolic Ca2+ that peaked within 3 min using FURA-2AM fluorescence; an event not observed when cells were incubated with 10−8 M dexamethasone (DEXA). Consistent with these results, incubation with 10−8 M ALDO led to increases in the oxidative-respiratory burst [superoxide production] (P<0.01, n=3); an event not observed when cells were incubated with either 10−8 or 10−7 M dexamethasone. The oxidative responses to ALDO were blunted by pre-incubation of cells with 1 uM canrenoic acid (CA), a well-described MR antagonist (P<0.03, n=3). We then studied the effect of ALDO on HL-60 transmigration and observed that 2 hr incubation at 37C with 10−8 M ALDO led to augmented migration (P<0.03, n=2) when compared to vehicle as estimated by CyQuant cell migration assays. We then isolated untouched circulating human neutrophils by immunomagnetic isolation following density gradient sedimentation with PolymorphPrep from otherwise healthy subjects. Flow cytometric analyses showed greater than 97% neutrophils as these cells were positive for CD45, CD16 and CD66b. Live/dead cell automated analyses shows greater than 90% cell viability by acridine orange and propidium iodide fluorescence. These cells likewise express MR as determined by western blot analyses for MR as reported in kidney and endothelial cells. Cells incubated with ALDO (10−8 M) showed a rise in cytosolic Ca2+ and an increase in the oxidative-respiratory burst (P<0.01, n=3); a response that was sensitive to 1 uM CA. We also observed that 4 hr 10−9M ALDO incubation led to augmented neutrophil transmigration (P<0.03, n=2). Thus our results suggest that activation of MR by ALDO leads to neutrophil activation that may contribute to the inflammatory responses associated with MR activation in vivo. Disclosures: No relevant conflicts of interest to declare.

Discontinuation of Imatinib Therapy in Chronic Myeloid Leukemia Patients with Sustained Complete Molecular Response4.5 (CMR4.5)

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2763-2763
Author(s):  
Hyun-Gyung Goh ◽  
Soo-Young Choi ◽  
Ju-Hee Bang ◽  
Soo-Hyun Kim ◽  
Eun-Jung Jang ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Digital Pcr ◽  
Imatinib Therapy ◽  
Risk Scores ◽  
Accurate Estimation ◽  
Molecular Response ◽  
First Line ◽  
Pcr Assay ◽  
Transcript Levels

Abstract Abstract 2763 Approximately 50% of CP CML patients achieve complete molecular response (CMR) at 6–7 years of first-line imatinib therapy. Although imatinib therapy is effective in CML patients and a substantial portion of patients achieve CMR with prolonged imatinib therapy, up to 10^7 leukemic cells can still be present in the absence of detectable BCR-ABL in RQ-PCR assay due to the sensitivity limit of current RQ-PCR technology. The recent data from STIM (Stop Imatinib) trial showed that the probability of persistent CMR at 12 month follow-up after imatinib discontinuation was 41%, and the conclusion was that imatinib can be safely discontinued, at least in some patients with persistent CMR. However, it is still not clearly defined whether discontinuation of imatinib therapy can be safely employed in patients with sustained CMR. In our prospective study, we examined if imatinib therapy can be safely discontinued in CML patients with sustained CMR4.5 according to strict PCR sensitivity criteria, and CMR4.5 was defined as undetectable BCR-ABL using RQ-PCR assay with at least 4.5-log sensitivity. CML patients who were treated with imatinib for more than 3 years and whose BCR-ABL was undetectable in RQ-PCR for at least 2 years were enrolled in this study. Our primary objectives were to evaluate the probability of persistent CMR4.5 at 12 month follow-up after discontinuation, and to measure the duration of persistent CMR4.5 after discontinuation. The secondary objective was to evaluate the probability of major molecular response (MMR) loss and the time taken to lose MMR at 12 month follow-up after discontinuation. In patients with loss of MMR, the probability of re-achieving MMR/CMR4.5 and the time taken to re-achieve MMR/CMR4.5 after imatinib resumption were also evaluated. After discontinuation, molecular response was monitored using RQ-PCR assay every month up to 6 month follow-up, every 2 months up to 12 month follow-up, and every 3 months thereafter. Digital PCR methodology with higher sensitivity compared to RQ-PCR assay was also applied before discontinuation and every year after discontinuation for more accurate estimation of BCR-ABL transcript levels. In case of relapse, defined as loss of MMR on 2 consecutive assessments, imatinib therapy was re-introduced and molecular response after resumption was observed using both RQ-PCR and digital PCR assays. As of data cut-off date of 15 Jul 2011, 20 patients (13 females, 7 males) who were diagnosed in Seoul St. Mary's Hospital between 20 Mar 1996 and 25 Apr 2005 were enrolled in this study with a median follow-up of 7 months (range, 2–9), and informed consents were obtained from all patients prior to participation. With a median age of 44 years (range, 25–67), the percentages of patients with low, intermediate and high Sokal risk scores were 30%, 30% and 15%, respectively with unknown Sokal risk scores in 25%. Ten patients (50%) received SCT and/or interferon therapy prior to imatinib therapy, while 10 patients (50%) received first-line imatinib therapy. The median time on imatinib therapy and the median duration of sustained CMR4.5 were 91 months (range, 40–112) and 60 months (range, 23–104), respectively, prior to discontinuation. Since discontinuation of imatinib therapy, all of 20 patients remained off therapy at the last follow-up with persistent CMR4.5 in 18 patients (90%) and loss of CMR in 2 patients (10%). Although loss of CMR was observed in 2 patients, both patients have not resumed imatinib therapy as MMR was maintained at the last follow-up. Our preliminary data show lower relapse rate after discontinuation compared to previous discontinuation studies. Strict PCR sensitivity criteria should be employed to assess the accurate measurement of BCR-ABL transcript levels prior to discontinuation, and then it might be possible to safely stop imatinib therapy in CML patients with stable CMR4.5. Through further clinical investigation on a large patient population and longer period of observation, more concrete conclusion can be made regarding the outcome of imatinib discontinuation. Updated data with longer follow-up duration will be presented in the meeting. Disclosures: No relevant conflicts of interest to declare.

The Predictive Value of Early Molecular Response in Chronic Phase CML Patients Treated with Dasatinib First Line Therapy

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 785-785 ◽  
Author(s):  
David Marin ◽  
Corinne Hedgley ◽  
Richard E Clark ◽  
Jane F Apperley ◽  
Letizia Foroni ◽  
...  
Keyword(s):  
Peripheral Blood ◽  
Predictive Value ◽  
Conflicts Of Interest ◽  
Chronic Phase ◽  
Transcript Level ◽  
Molecular Response ◽  
Costal Margin ◽  
Molecular Responses ◽  
Transcript Levels ◽  
International Scale

Abstract Abstract 785 We assessed the correlation between molecular response at 3 and 6 months of dasatinib 100mg daily treatment and subsequent cytogenetic and molecular responses in 150 newly-diagnosed chronic phase CML patients treated with front line dasatinib in the UK SPIRIT 2 study (imatinib vs dasatinib). The median age was 54 years (range 18.4–82.1); 90 patients were male. The Sokal risk distribution was: 39 low, 65 intermediate and 46 high. At diagnosis 26 patients had splenomegaly >10cm below the costal margin; median WBC and platelet count were 65.7 (2.2-428) and 404 (101-2,433). The median hemoglobin level was 11.0 g/dl (4.17-15.8). The median percentage of blasts and basophils in peripheral blood was 0.4% (0-13.5) and 3.6% (0-19.2) respectively. The dose of dasatinib was adjusted according to tolerance. BCR-ABL1 transcripts in the peripheral blood were analyzed at 12 week intervals using RQ-PCR. Results were expressed as percentage ratios relative to an ABL1 internal control and expressed on the international scale. Complete molecular response (CMR) was defined as two consecutive samples with no detectable transcripts (RQ-PCR negative) and ABL1 control >40,000 (the median ABL1 control in the CMR samples was 96,000). In addition, we also explored a less stringent definition of CMR, namely CMR4.5 which was recently defined by the EUTOS group as BCR-ABL1 ratio of 0.0032 on the international scale, consistent with a 4.5 log reduction in the transcript level, without necessarily being RQ-PCR negative. With a median follow up of 15 months (range 6–29) the 2 year cumulative incidences (CI) of CCyR, MMR, CMR4.5 and CMR were 84.5, 72.1, 24.1 and 5.6% respectively. The median BCR-ABL/ABL ratios at 3, 6, 12 and 24 months were 0.830%, 0.093%, 0.040% and 0.034% respectively. We investigated the predictive value of the BCR-ABL1 transcript levels at 3 (>10% vs ≤10% and >1% vs ≤1%) and 6 months (>1% vs ≤1%) of dasatinib therapy on the 2 years CI of cytogenetic and molecular responses. The 135 patients who at 3 months had a BCR-ABL1/ABL1 ratio ≤10% and the 81 patients who had a ratio ≤1% had a significantly better 2 year CI of CCyR (89.1% vs 50.2%, p=0.02 and 100% vs 84.7%, p=0.01), MMR (83.7% vs 14.2%, p=0.004 and 85.2% vs 54.3 p<0.001), CMR4.5 (25.0% vs 0%, p<0.18 and 37.6 v 3.3% p=0.001) but not CMR (6.7 vs 0%, p=0.51 and 7.1 vs 0% p=0.46). Similarly, the 109 patients who at 6 months had a transcript ratio ≤1% had a better 2 year CI of MMR (86.3 vs 13.9%, p<0.001), CMR4.5 (31.2 vs 0%, p=0.03) and CMR (14.3 vs 0%, p=0.04) than the remaining patients. We used a receiver operating characteristic (ROC) curve to identify the optimal cut-off in the transcript level at 3 and 6 months that would predict the probability of each outcome with maximal sensitivity and specificity. Table 1 shows the results of applying the optimal cutoffs for each outcome in the 3-month analysis. Then we investigated whether the various outcomes could be better predicted using the cut-offs defined at 3 or at 6 months (including both the 1 and 10% cut-offs and the newly identified cut-offs) by using a multivariate model. For each outcome the cut-off defined at 3 months shown in Table 1 was superior. No pre-therapy patient characteristics were an independent predictor for cytogenetic or molecular response.Table 1.CI of the various responses according to cut-offs identified in the ROC analysis.3 month BCR-ABL1/ABL1 ratio (%)n2 year CI (%)pCCyR10493.3p<0.001≤2.724675.9>2.72MMR7987.6p<0.001≤0.967152.7≥0.96CMR4.55649.7p<0.001≤0.378947.1>0.387CMR4220.1p=0.01≤0.241080>0.24 The key finding from this analysis is that patients who achieve a transcript level ≤10% after 3 months of dasatinib (135 of 150) have an 89.1% probability of eventually achieving CCyR, compared to 50.2% for patients with higher transcript levels (p=0.02). This preliminary observation may allow the identification of around 10% of dasatinib-treated patients for whom other forms of treatment might be considered although our conclusions require verification in further studies. The predictive power of RQ-PCR assessment can be greatly improved by identifying the optimal cut-offs for the specific outcomes, which is particularly important when predicting for the achievement of CMR. It remains uncertain whether these differences in response will translate into differences in survival and the SPIRIT 2 study continues to address this question. Disclosures: No relevant conflicts of interest to declare.

MiR-125b Cooperates with BCR-ABL and Enhances Therapy Resistance in Childhood Ph+ ALL

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 78-78
Author(s):  
Marina Lipkin Vasquez ◽  
Nicola Zanesi ◽  
Stefano Volinia ◽  
Ramiro Garzon ◽  
Giovanni Cazzaniga ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Blast Crisis ◽  
Therapy Response ◽  
Therapy Resistance ◽  
Poor Responder ◽  
Intrathecal Methotrexate ◽  
Patient Groups

Abstract Abstract 78 The treatment improvements in the past two decades led childhood acute lymphoblastic leukemia (ALL) cure rates reach over 80%, but children who carry the Philadelphia (Ph+) chromosome still have high risk of relapse due to therapy resistance. Different results with large series of patients have shown that an earlier remission after induction with glucocorticoids and intrathecal methotrexate is correlated to a better outcome. Besides, the minimal residual disease (MRD) risk measured after induction is also correlated to the therapy response, indicating that an early response can predict Ph+ ALL overall outcome. Several studies have shown that Ph+ ALL is heterogeneous in terms of clinical parameters even if all patients carry BCR-ABL and most of them carry the oncogenic isoform of IKAROS. They respond differently to the treatment, which suggests the presence of additional mechanisms involved in leukemogenesis. In an attempt to find secondary genetic abnormalities that may be responsible for the Ph+ ALL chemo resistance and heterogeneity, we studied the miRNA expression profile in two patient groups discriminated by the initial therapy response. We included samples from 78 consecutive Ph+ ALL children diagnosed in Italy between 2000 and 2010. The miRNA signature was analyzed by miRNA array using Applied Biosystems Array-Cards comparing two patient groups differentiated according to MRD and prednisone response. A particular miRNA profile was found in the poor responder group and it was confirmed using specific miRNA single assays from AB. Specially the miR-125b was up to ten-fold overexpressed compared to the good responder group (p =.006). To investigate the functional role of miR-125b in Ph+ ALL we used BCR-ABL positive cell lines (3 ALL and one CML in blast-crisis) to create a xenograft leukemia model and induced miRNA upregulation by direct inoculation of synthetic miR-125b (premiR). Tumors injected with premiR-125b (n=14) were significantly bigger than the scrambled oligonucleotide (n=10) and mock controls (n=4) (p=.04). After two weeks the premiR-125b tumors grew six-fold more than controls and average tumor weights for the scrambled oligonucleotides and the premiR-125b inoculated mice were 0.63 g and 1.56 g, respectively (p =.01). Further, we determined whether miR-125b protects the tumor cells from apoptosis after treatment with Dexamethasone. While tumors injected with scrambled molecules were 70% reduced after 10 days of corticoid treatment, tumors injected with premiR-125b were only 30% reduced (p=.05). Together, these results suggest that miR-125b acts as an oncogene in Ph+ ALL and its overexpression accelerates the role of BCR-ABL, increasing therapy resistance and leukemia aggressiveness. Additional studies will be necessary to understand why this miRNA is overexpressed in some patients and how it acts in cooperation with BCR-ABL to induce leukemia. In the future, novel therapies using miRNAs as targets can emerge as strategies to be added to the anti-tyrosine kinase drugs in order to improve treatment response and survivor in Ph+ ALL. Disclosures: No relevant conflicts of interest to declare.

Homoharringtonine Contributes to Imatinib Sensitivity by Blocking EphB4/RhoA Pathway in Ph+ Myeloid Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4420-4420
Author(s):  
Liu Xiaoli ◽  
Bintao Huang ◽  
Qingfeng Du ◽  
Jinfang Zhang ◽  
Na Xu ◽  
...  
Keyword(s):  
Western Blot ◽  
Cell Viability ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Annexin V ◽  
Apoptosis Rate ◽  
Xenograft Models ◽  
Stimulation Of ◽  
In Cells

Abstract Abstract 4420 Objective: The purpose was to investigate the role of the EphB4 in imatinib (IM) resistance and the mechanism why the homoharringtonine (HHT)+IM regimen gained more treatment profits than simple HHT or IM treating myeloid leukemia. Method: The stable under-expressing EphB4 cells (K562-R-EphB4-sh) were obtained. The cell viability and IC50 under the incubation of IM or HHT+IM was tested by MTT. PE Annexin V apoptosis detected the apoptosis rate of K562-R cells. Subcutaneous K562 xenograft models were established. The activated signal proteins in cells and tissues such as RhoA, MEK and ERK were tested by Western blot. Result: K562-R-EphB4-sh cell and xenograft were sensitivity to IM. Activated RhoA was not involved in K562-R-EphB4-sh cell and xenograft tissue. But phosphorylation of MEK/ERK was overexpression in K562-R-EphB4-sh cell and tissue. The apoptosis rate reached 58.71 ± 2.39% with K562-R cell incubated with HHT+IM, which was higher to K562-R cell incubated with IM (P=0.002). IC50 of K562-R cell incubated by IM was 5.45 mg/L. But under the stimulation of HHT+IM, IC50 of K562-R decreased from 5.45 to 1.17 mg/L (P<0.001). K562-R xenograft volumes significantly decreased with IM+HHT treatment comparing with before treatment (1692.82 ± 317.14 mm3 versus 975.56 ± 132.42 mm3, P<0.001). HHT blocked the expressions of EphB4/RhoA in K562-R cell and xenograft, but HHT cannot down-regulate the expression of P- MEK/ERK. Conclusions: A new marker of IM resistance mediated by the activation of EphB4/RhoA pathway. HHT+IM regimen gained more treatment profits than simple HHT or IM treating myeloid leukemia by blocking EphB4/RhoA pathway in Ph+ myeloid leukemia. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Src-like Adaptor Protein Promotes the CD103+ dendritic Cell Homeostasis in the Lung

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1339-1339
Author(s):  
Namit Sharma ◽  
Pan Zhongda ◽  
Tracy Lauren Smith ◽  
Savar Kaul ◽  
Emilie Ernoult ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Immune Response ◽  
Immune Cell ◽  
Conflicts Of Interest ◽  
Adaptor Protein ◽  
Cell Receptor ◽  
Apoptotic Cells ◽  
Direct Role

Abstract Dendritic cells (DCs) along with mast cells function as sentinels for the innate immune system and perform as antigen presenting cells (APCs) to mount an adaptive immune response against invading pathogen. FLT3 receptor tyrosine kinase signaling has been shown to regulate the homeostatic mechanisms of subsets of DCs particularly, CD103+DCs compared to CD11b+DCs. CD103+DCs are regarded as APCs with superior capabilities to mount an effective immune response, thus understanding their homeostasis mechanism(s)/function is of paramount importance to devise effective therapeutics including DC vaccines. The Src-like adapter protein (SLAP) has been shown to dampen the signaling downstream of receptor tyrosine kinases including FLT3, cKit, and immune cell receptors including T cell receptor, B cell receptor, and Granulocyte-monocyte colony stimulating factor receptor via by recruiting c-Cbl, an ubiquitin ligase. Here, we report that SLAP deficient mice (KO) have reduced numbers of CD103+DC in lung while equal numbers in liver and kidney compared to control mice. To further confirm reduced CD103+DC in the lung, efferocytosis assays that are dependent upon CD+103 DC in lung epithelium to cleanse the apoptotic cells were performed. Flow cytometric quantification of CD103+DCs that uptake fluorescently labeled apoptotic cells administered via intranasal route and migrate to mediastinal lymph nodes confirmed reduced number of CD103+DCs in SLAP KO mice. Further analysis of DC progenitor populations showed reduced pre-DC progenitor in the lung in SLAP KO mice while bone marrow compartment showed equal progenitor populations including pre-DC and common dendritic progenitors suggesting the role of SLAP in localized FLT3 signaling in the lung. Consistently, DCs in lymphoid compartment including spleen, thymus, inguinal and popliteal lymph node did not show any defects. Upon further dissecting the cellular mechanism, SLAP KO DCs showed increased apoptosis while having similar proliferation potential in vivo at steady state.Bone marrow progenitors from SLAP KO mice failed to generate mature DCs in the presence of FLT3 ligand in vitrodue to enhanced apoptosis at early time points. Also, submaximal inhibition of FLT3 with an inhibitor, quizartinib partially rescues the apoptotic phenotype of SLAP KO bone marrow progenitors suggesting a cell-intrinsic role of SLAP in the survival of DCs. Biochemical analysis revealed that SLAP is directly recruited to the juxta-membrane residues of the FLT3 receptor in an inducible manner suggesting a direct role of SLAP in the regulation of FLT3 signaling. Phosphoflow analysis of DCs generated in the combined presence of GMCSF and FLT3 ligands showed that SLAP promotes the signaling to SHP2 while perturbs signaling to the mTOR pathway. Together these results suggest that SLAP is a critical regulator of CD103+DCs homeostasis in selective peripheral organs including the lung. Disclosures No relevant conflicts of interest to declare.

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