FLT3 TKI Are Differentially Affected By Both Plasma Binding and Stromal Survival Factors

Abstract The FMS-like tyrosine kinase 3 (FLT3) is the most frequently mutated gene in acute myeloid leukemia (AML). As such it represents an attractive target for tyrosine kinase inhibitors (TKI) for the treatment of AML, a number of which have entered clinical trials. However, to date, these trials have demonstrated limited clinical efficacy. Evidence indicates that failure to achieved adequate inhibitory activity against FLT3, at least partially as a result of plasma protein binding, as well as survival factors provided by stroma, are among the reasons for the limited efficacy of FLT3 TKI. In this study we have characterized the effects of plasma binding and stromal survival factors on TTT-3002, a novel TKI that has demonstrated potent inhibition of the internal tandem duplication (FLT3-ITD) as well as a large panel of clinically described kinase mutants (FLT3-KD), and compared it to the effects these same conditions have on other FLT3 TKI. Using plasma from pediatric/young adult patients of a variety of ages (n = 24, median 7.2 years, 4.1-36.9 years), the impact of human plasma binding on the activity of TTT-3002 against FLT3-ITD dependent proliferation. 50% human plasma results in a moderate decrease in activity of TTT-3002 (IC50 45.2 nM in human plasma, 2.4 nM in media with 10% FCS). This is a markedly smaller shift than several TKIs currently or previously in clinical trials including sorafenib, CEP-701, PKC-412, and AC-220 (50 to >500-fold increase in IC50). The data demonstrate a clear linear relationship between plasma alpha-1 acidglycoprotein (AGP), an acute-phase reactant, and fold-increase in IC50 of TTT-3002 (1.7 fold-increase per 10 mg/dL AGP). The data also demonstrate a wide variability in patient plasma AGP concentration that is dependent upon age, but also appears to be significantly impacted by position in treatment (median 117 mg/dL, 58-354 mg/dL). Plasma AGP strongly inhibits several FLT3 inhibitors including CEP-701 and PKC-412 (>100-fold increase in IC50); whereas other FLT3 inhibitors such as sorafenib and AC-220 are inhibited by human plasma in an AGP-independent manner. These data indicate the presence of several human plasma proteins whose binding leads to drug inhibition that TTT-3002 is nonetheless resistant to, in comparison to other clinically relevant FLT3 inhibitors. The identification of these proteins is ongoing. Using primary human bone marrow stromal cell co-culture with two FLT3-ITD cell lines increases the IC50 of TTT-3002 but to a lower extent than that of AC-220 (MV4-11: 1.1-1.9 fold versus 2.3-2.7 fold, MOLM-14: 2.2-2.3 fold versus 3.5-3.6 fold). The data are consistent across stromal cells cultured from several different pediatric patients (n = 5). These data suggest that TTT-3002 may be more resistant to some of the inhibitory effects that have led to disappointing results with other similar FLT3-targeted TKIs. As such, TTT-3002 represents an appealing candidate for clinical studies. Disclosures No relevant conflicts of interest to declare.

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Human Genetic Diversity Alters Therapeutic Gene Editing Off-Target Outcomes

Blood ◽

10.1182/blood-2021-152429 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3993-3993

Author(s):

Linda Yingqi Lin ◽

Samuele Cancellieri ◽

Jing Zeng ◽

Francesco Masillo ◽

My Anh Nguyen ◽

...

Keyword(s):

Genetic Diversity ◽

Clinical Trials ◽

Genome Editing ◽

Gene Editing ◽

Conflicts Of Interest ◽

African Ancestry ◽

Great Promise ◽

Personal Genome ◽

Hematopoietic Stem ◽

The Impact

Abstract CRISPR gene editing holds great promise to modify somatic genomes to ameliorate disease. In silico prediction of homologous sites coupled with biochemical evaluation of possible genomic off-targets may predict genotoxicity risk of individual gene editing reagents. However, standard computational and biochemical methods focus on reference genomes and do not consider the impact of genetic diversity on off-target potential. Here we developed a web application called CRISPRme that explicitly and efficiently integrates human genetic variant datasets with orthogonal genomic annotations to predict and prioritize off-target sites at scale. The method considers both single-nucleotide variants (SNVs) and indels, accounts for bona fide haplotypes, accepts spacer:protospacer mismatches and bulges, and is suitable for personal genome analyses. We tested the tool with a guide RNA (gRNA) targeting the BCL11A erythroid enhancer that has shown therapeutic promise in clinical trials for sickle cell disease (SCD) and β-thalassemia (Frangoul et al. NEJM 2021). We find that the top predicted off-target site is produced by a non-reference allele common in African-ancestry populations (rs114518452, minor allele frequency (MAF) = 4.5%) that introduces a protospacer adjacent motif (PAM) for SpCas9. We validate that SpCas9 generates indels (~9.6% frequency) and chr2 pericentric inversions in a strictly allele-specific manner in edited CD34+ hematopoietic stem/progenitor cells (HSPCs), although a high-fidelity Cas9 variant mitigates this off-target. This report illustrates how population and private genetic variants should be considered as modifiers of genome editing outcomes. We expect that variant-aware off-target assessment will be required for therapeutic genome editing efforts going forward, including both ongoing and future clinical trials, and we provide a powerful approach for comprehensive off-target prediction. CRISPRme is available at crisprme.di.univr.it. Disclosures No relevant conflicts of interest to declare.

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Circulating CLL Cells Expressing CD49d Display a Phospho-Proteomic Profile Consistent with a Constitutive Receptor Engagement by Blood-Borne Ligands

Blood ◽

10.1182/blood.v120.21.930.930 ◽

2012 ◽

Vol 120 (21) ◽

pp. 930-930

Author(s):

Dania Benedetti ◽

Erika Tissino ◽

Daniela Marconi ◽

Michele Dal Bo ◽

Riccardo Bomben ◽

...

Keyword(s):

Cell Growth ◽

Tyrosine Kinase ◽

Translational Control ◽

Conflicts Of Interest ◽

Control Cell ◽

Adaptor Protein ◽

Fold Increase ◽

Lymphocytic Leukemia ◽

Strong Increase ◽

Lim Kinase

Abstract Abstract 930 Chronic lymphocytic leukemia (CLL) cell growth and survival occur in defined microenvironment niches controlled by several receptor/ligand interactions including those mediated by the α4β1 integrin. This integrin, in particular, is known to interact with both the extracellular matrix component fibronectin, via the non-RGD CS-1 fragments, and the bone marrow/stromal components, via the vascular-cell adhesion molecule-1 (VCAM-1). The α4 integrin chain (a.k.a. CD49d) has been previously shown to be associated with poor prognosis in CLL by marking a subset of CLL patients characterized by a more aggressive clinical course both in term of disease progression and overall survival. Despite the great deal of studies investigating CLL cell microenvironmental interactions in tissue sites, little is known regarding the constitutive engagement of adhesion receptors in circulating CLL cells, and the role in this context of plasma/plasma components. To address this issue, the proteomic profiles of circulating leukemic cells from 80 CLL cases expressing or not CD49d were explored using a reverse phase protein microarray (RPMA) approach quantitatively analysing 77 proteins (61 phosphoproteins) known to be involved in translational control, cell growth, apoptosis, B cell receptor and cytoskeletal signaling. Comparison of the signaling activation portrait between 40 CD49d pos and 40 CD49d neg CLL cases highlighted the over-expression in CD49d pos CLL of proteins involved in the regulation of integrin-mediated cytoskeletal dynamics, such as phospho-p21-activated kinase (PAK1 Ser199-204/PAK2 Ser192-197; p=0.0005), phospho-LIM kinase (LIMK1 Thr508/LIMK2 Thr505; p=0.00001) and the adaptor protein CrkII Tyr221 (p=0.039). Moreover, CD49d pos CLL cells were characterized by a high correlation between proteins involved in integrin-mediated signal trasduction, including the focal-adhesion kinase (FAK Tyr576-Ser577), the tyrosine kinase Src Tyr527 and the adaptor protein CrkL Tyr207. Since PAK and LIMK represent key players in the modulation of the structure and the activity of actin cytoskeleton, we focused on these proteins for validation experiments. The constitutive pPAK and pLIMK overexpression in the CD49d pos CLL group was confirmed by western blot analysis comparing purified CLL cells from 3 CD49d pos versus 3 CD49d neg cases (mean fold increase >100 for both proteins). The results obtained suggest that integrin signalling is constitutively active in CD49d-expressing circulating CLL cells, pointing to a constitutive receptor engagement by CD49d ligands allegedly present in plasma. To test whether plasma constituents could modulate integrin-signaling proteins, CLL cells from 5 cases, expressing or not CD49d, were challenged with autologous plasma (1:3 dilution) for 30 seconds, 1 and 3 minutes. The presence of plasma induced a strong increase of PAK and LIMK phosphorylation intensities in CD49d pos CLL cells, starting at 30 seconds upon stimulation (mean fold increase >10 as compared to control for both proteins), and increasing after 1 and 3 minutes (mean fold increase >40 and >50 for both the proteins, respectively). Conversely, plasma stimulation did not induce pPAK and pLIMK expression modulation in CD49d neg CLL cells. Of note, pretreatment of CD49d-expressing CLL cells with the anti-CD49d HP1/2 blocking antibody, resulted in lower up-regulation of pPAK and pLIMK with an overall 60% inhibition for both the proteins (p=0.01), confirming the involvement of CD49d triggering in the observed activated signaling. Given the above results, we investigated plasma from 24 CLL patients, expressing CD49d at different levels, for the presence of the CD49d ligand fibronectin using an ELISA detection kit. All samples tested showed more than 1 mg/ml fibronectin concentration, without differences between CD49d-expressing and CD49d neg CLL cases. Altogether these results sustain the hypothesis of an active role of plasma/plasma components in the activation of CD49d-mediated integrin pathway, thus favoring the delivery of chemoresistance/pro-survival signals even in the context of circulating CLL cells. Our results may be of interest in the perspective of novel therapies (e.g. Bruton tyrosine kinase inhibitors) known to provoke a massive egress of neoplastic cells from tissue sites into the blood stream. Disclosures: No relevant conflicts of interest to declare.

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Significant Increase in Reporting of Transfusion Reactions with the Implementation of an Electronic Reporting System

Blood ◽

10.1182/blood.v126.23.4740.4740 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4740-4740 ◽

Cited By ~ 1

Author(s):

Rosanne St. Bernard ◽

Matthew Yan ◽

Shuoyan Ning ◽

Alioska Escorcia ◽

Jacob M Pendergrast ◽

...

Keyword(s):

Reporting System ◽

Conflicts Of Interest ◽

Population Level ◽

Fold Increase ◽

Medical Director ◽

Care Hospital ◽

Transfusion Reaction ◽

Reaction Reporting ◽

Electronic Reporting ◽

The Impact

Abstract Introduction In robust hospital transfusion services, transfusion reaction reporting triggers a structured response to the assessment, diagnosis, and clinical management of the individual. At a population level, the feedback loop of hemovigilance permits the perception of signals applicable to donors, material production, patterns of use, infusion care, and recipient vulnerabilities. Transfusion reaction reporting therefore aims to improve the quality of patient care and safety in transfusion, which remains one of the most commonly performed procedures in medicine today. Large variations between passive/retrospective or active/prospective systems imply underreporting. Reasons for this may lie in unawareness of, nihilism on, or obstacles to this duty. In 2009, our center transitioned from a paper-based to an electronic reporting system (ERS) for suspected patient reaction events (PREs). This study sought to determine the impact of this change on PRE reporting rates. Methods This study was conducted in Toronto, Canada at the University Health Network, a 4-site, 767-bed, ternary care hospital with high transfusion activity (2014: 60,000 component and 30,000 derivative dispensations). In 05/2009, hardcopy mountsheets for transfusion labels were revised to provide space for recording corresponding vital signs, with instructions on PRE reporting. Medical director PRE review followed with event documentation in a transfusion laboratory database (recording imputability, reaction type, severity, and implicated product(s)). An Acute Transfusion Reaction policy was also developed to protocolize and further streamline the approach to various reactions, but was not implemented across all sites until 11/2009. At this time, electronic PRE reporting went live in the existing electronic medical record, with medical director review hereafter culminating in uploaded case conclusions. Technical tutorials on healthcare worker reporting spanned several months before implementation, without emphasizing the theory or evidence-based value of hemovigilance. The quantity and characteristics of reactions pre-/post-ERS implementation were compared. Results Prior to the ERS option (5/2009-11/2009), the reported PRE rate was 0.26/day. Subsequent to launch (11/2009-12/2009), the reported PRE rate was 0.66/day, representing a 2.54 fold increase (p<0.05) (Figure 1). This nearly-trebled rate has been sustained throughout subsequent years: 01/2010-12/2010: 0.88/day; 01/2011-12/2011: 0.87/day; 01/2012-12/2012: 0.87/day; 01/2013-12/2013: 0.88/day; 01/2014-12/2014: 0.93/day. The distribution of PRE conclusions pre-ERS was: febrile non-hemolytic transfusion reaction (FNHTR) 40%; unrelated to transfusion (UTR) 34%; allergic transfusion reaction (ATR) 7.3%; query bacterial contamination (BaCON) 4.9%; transfusion related acute lung injury (TRALI) 3.6% and transfusion related circulatory overload (TACO) 2.4%. The distribution of PRE conclusions after ERS (11/2009-12/2014) was: ATR 28.8%; UTR 28.7%; FNHTR 19.9%; TACO 8.6%; transfusion associated dyspnea 4.7%; pain 3.2%; query BaCON 2.3% and TRALI 1.7%. Conclusions Our data demonstrate that PRE reporting significantly increased and was sustained after the implementation of an ERS. This finding suggests that despite a dearth of strategies to address underreporting, the solution may lie in removing disincentives while facilitating action in familiar practice platforms. Two other studies investigated the implementation of an ERS for transfusion reaction reporting (Fujihara H. et al. 2015; Yeh, S et al. 2011), with one confounded by a significant increase in transfusion rates in the post-ERS period. In contrast, our denominator of blood utilization has been stable or decreasing across sites over the last five years, with 3% of product recipients nevertheless experiencing a PRE. Despite the significant increase in reported PREs, we did not see an increase in UTRs (34% vs 25%) to account for the difference, arguing against "junk inflations," while rather suggesting that reporter suspicions generally concur with specialist conclusions on transfusion imputability. Given the importance of accurate transfusion reaction reporting for patient safety, we suggest that this strategy be considered by other centers to improve reporting activity with its potential downstream benefits. Disclosures No relevant conflicts of interest to declare.

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Photochemical Pathogen Inactivation Treatment of Human Plasma (amotosalen + UVA) Has No Major Impact on the Protein expression pattern Assessed by a 2-DIGE Proteomic Assay.

Blood ◽

10.1182/blood.v112.11.1994.1994 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1994-1994 ◽

Cited By ~ 2

Author(s):

Jean-Pierre Cazenave ◽

Philippe Ohlmann ◽

Hervé Isola ◽

Christian Gachet

Keyword(s):

Clinical Trials ◽

Human Plasma ◽

Gel Electrophoresis ◽

Protein Modification ◽

Internal Standard ◽

2D Gel Electrophoresis ◽

Pathogen Inactivation ◽

2D Dige ◽

2D Gel ◽

The Impact

Abstract Background: Photochemical treatment (PCT) using amotosalen HCl and UVA light (3 J/cm2: 320 – 400 nm) inactivates pathogens and leukocytes in therapeutic single donor apheresis plasma prepared within 8 hr of collection (INTERCEPTTM [I-FFP], Cerus Europe, Amersfoort, Netherlands). Clinical trials demonstrated efficacy and safety supporting a Class III CE mark and AFSSAPS (Medicinal Products Agency of France) registration of I-FFP for primary therapeutic indications including: congenital and acquired coagulopathies and TTP. Although no evidence of immune response to neo-antigens has been detected in clinical trials (Transfusion2005; 45:1610) or in post marketing surveillance studies, it remains unknown to what extent plasma proteins may be altered by PCT. We measured the impact of PCT on plasma protein profiles using a quantitative and qualitative proteomic method (2D-DIGE) to further define the possible occurrence of protein modifications in I-FFP. Methods: Plasma units (650 ml) from 8 donors (4 male and 4 female) were collected by apheresis (Haemonetics MCS+, Braintree, MA) with AB16 anticoagulant. Plasma from each donor was separated into two plasma units, one was treated with the INTERCEPTTM system within 8 hr after collection, and the paired untreated unit served as a Control. The proteomes of I-FFP and Control plasma were evaluated using differential two-dimensional in-gel electrophoresis (2D-DIGE). Before fluorescent cyanine (Cy) labeling and 2D-gel electrophoresis, albumin and IgG, the more abundant proteins, were removed from plasma using a depletion kit (GE Healthcare). Prior to 2D gel electrophoresis, proteins from Control and I-FFP were labeled with fluorescent Cy3 (green) and Cy5 (red), respectively, in four samples and vice versa in the other four. Fifteen μg of protein from Control and or I-FFP plasma from each donor were mixed and resolved on the same minigel (7x7 cm). An internal standard (15 μg), labeled with Cy2 (blue), composed of equal amounts of plasma from each donor, was added in all experiment as a normalizing agent to monitor spot intensity for determination of reproducible quantitative differences of statistical significance (ANOVA, p < 0.05). After electrophoresis, images of 2D gels were generated using a fluorescent scanner and data were analyzed using the Samespots software (NonlinearTM). Results: 281 common protein spots were observed in the proteome pattern of plasma from all 8 independent experiments and included in the analysis. INTERCEPTTM treatment did not change any of these 281 spots (ANOVA, p > 0.05). Using the internal standard to monitor quantitative changes, more than 90% of protein spots demonstrated an expression difference of less than 10% while the highest change observed was 1.8 fold. Conclusion: Within 8 hrs after collection, pathogen inactivation of human plasma by the INTERCEPTTM process did not show evidence of plasma protein modification either in a qualitative or a quantitative manner. Further studies would be required to assess in greater detail the impact on protein modification of the process.

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Regulatory T Cells of G-CSF Mobilized Stem Cell Donors Qualify for Clinical Applications,

Blood ◽

10.1182/blood.v118.21.4055.4055 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4055-4055

Author(s):

Sya N. Ukena ◽

Sarvari Velaga ◽

Goudeva Lilia ◽

Philipp Ivanyi ◽

Arnold Ganser ◽

...

Keyword(s):

Clinical Trials ◽

T Cells ◽

Stem Cell ◽

Regulatory T Cells ◽

Conflicts Of Interest ◽

Interleukin 2 ◽

Fold Increase ◽

Hematopoietic Stem ◽

Cell Numbers

Abstract Abstract 4055 Recent clinical studies demonstrate the high potency of human regulatory T cells (Tregs) to control graft-versus-host disease following hematopoietic stem cell transplantation (SCT). Isolation of Tregs after recombinant G-CSF induced mobilization of stem cells would simplify the design of clinical trials in allogeneic SCT. However, there is growing evidence that G-CSF also exerts profound immune modulatory effects in the adaptive immune system. Therefore, we analyzed Tregs isolated by FACS based cell sorting from stem cell donors before (n=8) and after (n=13) G-CSF administration regarding their phenotype, stability, functional and expansion properties. Absolute CD4+ T cell (3.2 fold increase of mean after G-CSF; p<0.05) and CD4+CD25hi Treg cell numbers (4.1 fold increase of mean after G-CSF; p<0.01) were significantly increased after G-CSF administration. Analysis of the Foxp3 TSDR demethylation level displays a stable Foxp3 phenotype of G-CSF encountered Tregs (mean value 97.1% vs. 95.0 % after G-CSF administration). Moreover, the CD4+CD25hi Tregs of G-CSF treated SC donors suppress the proliferation of effector T cells with no significant differences to Tregs isolated from healthy donors before G-CSF treatment (mean values 42.1% vs. 49.9% after G-CSF administration at a Treg/ T effector cell ratio of 1:1). In vitro expansion of Tregs isolated after G-CSF application with anti-CD3 and anti-CD28 dynabeads in the presence of interleukin-2 led to comparable cell numbers as for the stimulation of control Treg cells. However, differences could be detected for the thymic derived marker molecule CD31 and those associated with activation (LAP, CD69, CD62L) and migration (CxCR3) as detected by FACS analysis. Our results show no significant differences regarding suppressive function and stability of Tregs isolated from stem cell donors before and after G-CSF administration. These data support the application of G-CSF mobilized Tregs for clinical trials which in turn opens up new possibilities for the adoptive transfer of Tregs in HSCT. Disclosures: No relevant conflicts of interest to declare.

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TTT-3002 Is a Novel FLT3 Tyrosine Kinase Inhibitor That Has the Potential to Overcome Some of the Limitations of Current FLT3 Inhibitors in Treatment of Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v120.21.866.866 ◽

2012 ◽

Vol 120 (21) ◽

pp. 866-866 ◽

Cited By ~ 1

Author(s):

Hayley S Wall ◽

Bao Nguyen ◽

Li Li ◽

Sarah M Greenblatt ◽

Patrick Brown ◽

...

Keyword(s):

Clinical Trials ◽

Tyrosine Kinase ◽

Progenitor Cells ◽

Myeloid Leukemia ◽

Kinase Inhibitor ◽

Free Drug ◽

Hematopoietic Stem ◽

Activating Mutations ◽

Flt3 Inhibitors

Abstract Abstract 866 Acute myeloid leukemia (AML) is a hematologic malignancy characterized by increased myeloproliferation and a block in differentiation of hematopoietic stem/progenitor cells, leading to infiltration of immature blasts in the bone marrow and peripheral blood. FMS-like tyrosine kinase-3 (FLT3) is a receptor tyrosine kinase expressed in hematopoietic stem/progenitor cells. It can activate cell growth/proliferation pathways via STAT5, AKT/PI3K, and RAS/MAPK signaling. Greater than 35% of AML patients harbor a constitutively activating mutation in the FLT3 gene, and the most common type, an internal tandem duplication (ITD), confers poor prognosis. These ITD mutations typically occur in the juxtamembrane domain and consist of variable length sequence repeats. In addition, activating mutations in the kinase domain are observed in 7–10% of patients. Thus, FLT3 is a validated target for the treatment of AML. Towards this end there have been a number of clinical trials testing the clinical efficacy of FLT3 tyrosine kinase inhibitors (TKIs) in FLT3 mutant AML, either alone or combined with chemotherapy. While some patients have responded, there have been many others who fail to demonstrate significant improvement. Additionally, many patients who initially respond to FLT3 TKI have developed resistance. Inadequate achievement of FLT3 inhibition has been one of the factors limiting efficacy in these trials. This appears most often to be due to a combination of insufficiently potent drugs, decreased efficacy against some FLT3 activating mutations, high plasma protein binding, and the selection for resistance-conferring point mutations within the FLT3/ITD gene. For all of these reasons, there is the need to develop additional FLT3 TKIs able to overcome some of these limitations. We report for the first time on TTT-3002, a kinase inhibitor that has activity against FLT3 and may overcome some of the limitations that current FLT3 TKIs have demonstrated in clinical trials. TTT-3002 is one of the most potent FLT3 inhibitors discovered to date with regard to both inhibition of FLT3 autophosphorylation and cell proliferation. Utilizing human FLT3/ITD mutant leukemia cell lines the half maximal inhibitory concentration (IC50) for inhibiting FLT3 autophosphorylation was less than 250 pM. The IC50 for TTT-3002 in inhibiting proliferation in these same FLT3/ITD expressing cells was 490–920 pM. TTT-3002 also showed potent activity when tested against a broad spectrum of known, non-ITD FLT3 activating mutations, including the most frequently occurring D835Y mutation. It also shows potent activity against a number of FLT3/ITD resistance mutations that have been selected for in patients from clinical trials of other FLT3 TKI or through in vitro drug screening. Studies utilizing human plasma samples from healthy donors and AML patients determined that TTT-3002 is only moderately protein bound, resulting in high levels of free drug able to bind target, and thus maintain activity, in the presence of physiological concentrations of major human plasma proteins including alpha-1-acid glycoprotein and human serum albumin. Therefore, relatively low levels of total drug would need to be achieved in vivo to achieve an effective concentration of free drug in clinical trials. These encouraging findings have been validated both ex vivo and in vivo utilizing mouse models of FLT3-associated AML. Survival and tumor burden of mice in a number of FLT3/ITD transplant models is significantly improved by administration of TTT-3002 via oral dosing. Finally, we demonstrate that TTT-3002 is cytotoxic to leukemic blasts isolated from FLT3/ITD expressing AML patients, while displaying minimal toxicity against normal hematopoietic stem/progenitor cells from healthy bone marrow donors. Therefore, TTT-3002 has demonstrated preclinical potential as a promising new FLT3 TKI that may overcome some of the limitations of other TKI in the treatment of FLT3-mutant AML. Disclosures: No relevant conflicts of interest to declare.

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The Impact Of Novel Splicing Abnormalities Of BCR-ABL On The Pathogenesis Of CML

Blood ◽

10.1182/blood.v122.21.5159.5159 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5159-5159

Author(s):

Junichiro Yuda ◽

Toshihiro Miyamoto ◽

Yoshikane Kikushige ◽

Jun Odawara ◽

Yasuyuki Ohkawa ◽

...

Keyword(s):

Tyrosine Kinase ◽

Conflicts Of Interest ◽

Kinase Domain ◽

Molecular Response ◽

Healthy Individuals ◽

Wild Type ◽

Abl Kinase ◽

Tki Treatment ◽

The Impact ◽

Splicing Patterns

Abstract Background Chronic myeloid leukemia (CML) is effectively treated with tyrosine kinase inhibitors (TKIs), but reactivation of BCR-ABL frequently occurs through acquisition of kinase domain point mutations. The mechanism of resistance in patients without BCR-ABL kinase domain point mutation is still elusive. Previous studies have revealed the abnormal splicing of BCR-ABL kinase domain, including Exon8/9 junction 35bp insertion and exon-skipping of Exon 7 (T O'Hare et al, Blood 2011. Gaillard JB, et al. Mol Cancer Ther 2010). The insertion of 35 intronic nucleotides at the exon 8/9 splice junction introduces a stop codon after 10 intron-encoded residues and inactive tyrosine kinase activity. The effect of these splicing abnormalities on susceptibility of cells against TKIs is still controversial. Furthermore, the conventional direct sequence techniques could not evaluate splicing abnormalities in major molecular response (MMR)- complete molecular response (CMR) CML patients, who achieved clinically leukemia-free state with a small number residual CML stem cells. Aims The aim of this study is to evaluate the frequency and the patterns of splicing abnormalities of BCR-ABL in CML patients, especially who achieved MMR-CMR by TKI treatment. Methods We analyzed peripheral blood samples from healthy individuals and CML chronic phase patients. We extracted total RNA from these samples and synthesized cDNA, and then performed PCR-amplification of BCR-ABL kinase domain in CML patients and ABL kinase domain in healthy individuals, respectively. PCR products were subjected to the amplicon sequence: We deeply sequenced BCR-ABL fusion gene transcripts, and evaluated splicing forms of BCR-ABL by using HiSeq 2000 (illumina). Results We successfully established a novel analysis method, which can detect the pattern of splicing abnormalities even in MMR-CMR patients. Using the amplicon sequence technique, we detected abnormal splicing patterns of BCR-ABL in 5 out of 15 CML patients. We also found that the splicing abnormalities were not restricted to 35bp insertion at the exon8/9 junction, thus intronic retention of intron 8 and intron 9 could be frequently detected with or without the 35bp insertion in CML patients (Table 1). Of note, these abnormal splicing patterns always co-existed with wild type BCR-ABL transcripts in all 5 cases analyzed. In addition to the novel splicing abnormalities in CML, we unexpectedly found in healthy individuals that splicing abnormalities such as 35bp insertion at the exon8/9 junction and intronic retention could be detected in ABL1 transcripts (Table 1). This result suggests that this sort of splicing abnormalities could occur at a certain frequency in steady state human hematpoiesis, and is not specific to BCR-ABL. Summary / Conclusion We have newly established an analysis system to efficiently detect splicing abnormalities of BCR-ABL even in MMR-CMR CML patients. Using this highly efficient amplicon sequence technique, we identified novel splicing abnormalities both in healthy individuals and CML patients, and found that the wild type BCR-ABL transcripts always co-exist with abnormally spliced BCR-ABL transcripts. These results collectively suggest that splicing abnormalities within the ABL1 kinase domain are not specific to CML patients treated with TKIs, and that the detection of such kinase domain splicing abnormalities do not reflect insusceptibility of the remaining cells during TKI treatment. Disclosures: No relevant conflicts of interest to declare.

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Non-Cycling Cells From Acute Myeloid Leukemia Patients Harbor the FLT3-ITD Mutation and Are Insensitive to TKI258, a Potent FLT3-Directed Inhibitor, in Vitro.

Blood ◽

10.1182/blood.v114.22.479.479 ◽

2009 ◽

Vol 114 (22) ◽

pp. 479-479

Author(s):

Caroline L Alvares ◽

Tino Schenk ◽

Sanna Hulkki ◽

Toon Min ◽

Gowri Vijayaraghavan ◽

...

Keyword(s):

Cell Expansion ◽

Conflicts Of Interest ◽

Leukemic Cell ◽

Trisomy 13 ◽

Leukemic Cells ◽

Multi Drug Resistance ◽

Hematopoietic Stem ◽

Flt3 Inhibitors ◽

The Impact

Abstract Abstract 479 The identification of activating mutations in the FLT3 gene and their impact on prognosis has been crucial to the rationale behind the development of FLT3 inhibitors. While it has been shown that some leukemic cells with high tumorigenic potential exist mostly in a dormant state, it is unclear if this quiescent/non-cycling leukemia-initiating fraction carries the FLT3-ITD mutation and if it is successfully targeted by FLT3 inhibitors. As a paradigm, quiescent Ph+ stem cells in CML have been shown to exhibit resistance to bcr-abl targeted inhibitors. Additionally, results from clinical trials suggest that FLT3 inhibitors reduce the leukemic blast count in peripheral blood but are less successful in the bone marrow where factors regulating hematopoietic stem cell quiescence are active. In order to investigate the non-cycling and cycling human leukemic cell boundary, we devised a biological model that allowed us to distinguish non-cycling AML cells from cycling AML progenitors in human FLT3-ITD positive AML samples. CD34+ cells were isolated from AML samples using magnetic cell sorting, labeled with the cell membrane dye PKH26 to enable tracking of cell division, and cultured on murine stroma for 12 days. Non-cycling AML cells were then separated from cycling cells by FACS sorting and were found to retain a CD34+ primitive phenotype in contrast to expanding leukemic blasts. Fluorescence in situ hybridization analyses revealed that non-cycling cells carried leukemic gene rearrangements (trisomy 8, trisomy 13, t[3;21]and t[16;16] in our cases), and were therefore part of the original leukemic clone. PCR for the FLT3-ITD region showed that in four out of five cases, the FLT3-ITD mutation was present in the non-cycling fraction. To examine the distribution of FLT3-ITD to FLT3 wild type (WT) bearing cells, non-cycling AML cells were FACS sorted, DNA extracted and the PCR products subsequently cloned. Bacterial colonies were sequenced and colony-PCR used to determine the ratio of FLT3-ITD to WT bearing colonies for each patient. These data indicated that at least 25% of non-cycling cells (range 25%-100%) harbored the FLT3-ITD mutation. We then assessed the impact of a potent FLT3-directed inhibitor, TKI258 (Novartis), on leukemic cell expansion and the viability of non-cycling cells. TKI258 has been found to induce apoptosis of FLT3-ITD bearing cells of the human acute monocytic leukemia MV4;11 cell line. In our present study, CD34+ AML blasts from the same FLT3-ITD positive patient samples were grown in vitro in the presence of 0 μM, 0.3 μM (IC50 dose) and 1.25 μM TKI258. In stromal cultures, TKI258 significantly reduced leukemic cell expansion (range 2.13 to 20 fold for untreated cultures and 0.07 to 2.27 fold for 1.25 μM TKI258 treated cultures at day 12, p ≤ 0.05). In methylcellulose colony assays, TKI258 exposure resulted in dose-dependent suppression of colony formation of CD34+ FLT3-ITD positive leukemia cells (60% to 81% reduction in the mean plating efficiency of CD34+ AML cells at 0.3 μM TKI258). Despite this striking anti-proliferative effect, the majority of non-cycling cells from AML patients showed resistance to TKI258 (five out of six cases). In these samples, FLT3-ITD positive non-cycling cells could still be detected after treatment with the equivalent highest clinical dose (1.25 μM) of TKI258. Moreover, at a functional level, limiting-dilution experiments on non-cycling AML cells pre-treated with TKI258 showed no impairment in a modified leukemic cobblestone assay at four weeks compared to untreated non-cycling cells. These results suggest that the majority of non-cycling AML cells that harbor FLT3-ITD are unaffected by a FLT3 inhibitor and may constitute an as yet untargeted disease reservoir. Only one FLT3-ITD AML case showed exquisite sensitivity to TKI258 with elimination of the non-cycling fraction observed from 0.3 μM of TKI258 upwards. Possible explanations for this may include specific mutant receptor sensitivities or generic multi-drug resistance mechanisms operating in dormant cells. Interestingly, TKI258 selectively eradicated an ‘intermediate' dividing progenitor population in two of the insensitive cases, an indication that leukemic progenitors may be rendered sensitive to FLT3 inhibition on transiting the dormancy-cycling boundary. Further studies are needed to determine if these findings are representative of a generation of FLT3 inhibitors or specific for TKI258. Disclosures: No relevant conflicts of interest to declare.

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Is There a Benefit of Adding Rituximab to CHOP in the Overall Survival of Patients with B-Cell Non-Hodgkin′s Lymphoma in a Developing Country ?

Blood ◽

10.1182/blood.v120.21.4892.4892 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4892-4892

Author(s):

Guillermo J. Ruiz-Delgado ◽

David Gomez Almaguer ◽

Luz C. Tarin-Arzaga ◽

Olga Cantú-Rodríguez ◽

Carlos Alarcon-Urdaneta ◽

...

Keyword(s):

Overall Survival ◽

B Cell ◽

Developing Country ◽

Conflicts Of Interest ◽

Positive Impact ◽

B Cell Lymphoma ◽

Fold Increase ◽

Developed Countries ◽

Number Of Cycles ◽

The Impact

Abstract Abstract 4892 Rituximab ( R ) has changed the prognosis of patients with non-Hodgkin′s lymphoma (NHL) in developed countries, but its role has not been analyzed in underprivileged circumstances. One hundred and two patients with NHL treated in a developing country were analyzed: 28 patients with follicular lymphoma (FL) and 74 diffuse large B cell lymphoma (DLCL). Patients were treated upfront with either CHOP or R-CHOP; the decision to employ R depending solely on the ability of patients to defray it. In DLCL, 42 were given CHOP and 32 R-CHOP, whereas in FL 19 were given CHOP and 9 R-CHOP. The impact of the addition of R was found to be more clear in FL than in DLCL. In patients with DLCL, the overall survival (OS) was 87% at 80 months for those treated with R-CHOP and 84% at 145 months for those treated with CHOP (p NS). In patients with FL, the OS was 89% at 88 months for those treated with R-CHOP and 71% at 92 months for those treated with CHOP (p =.05). In a multivariate analysis, other variables were identified to be associated with the OS were IPI and number of cycles in DLCL. It is concluded that rituximab produced a mild positive impact in the OS of patients with FL, but not in those with DLCL. Since the addition of rituximab results in a 36 fold increase in treatment costs, these observations may be important to decide therapeutic approaches in NHL patients living in underprivileged circumstances. Disclosures: No relevant conflicts of interest to declare.

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