Plasma Tyrosine Kinase Activity as A Potential Biomarker In BCR-ABL1-Targeted Therapy

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2738-2738
Author(s):  
Chen-Hsiung Yeh ◽  
Adam Abdool ◽  
Jean-marie Bruey
Keyword(s):  
Targeted Therapy ◽  
Tyrosine Kinase ◽  
Ex Vivo ◽  
Transcript Level ◽  
Microtiter Plate ◽  
Lymphocytic Leukemia ◽  
Myelogenous Leukemia ◽  
Serum Samples ◽  
Healthy Donors ◽  
Potential Biomarker

Abstract Abstract 2738 Introduction: In targeted therapy using tyrosine kinase (TK) inhibitors (TKIs), measurement of TK activity could help in diagnosis, identification of potential responders, and monitoring of treatment efficacy. Here we evaluated the feasibility of measuring circulating TK (cTK) activity in plasma in patients with BCR-ABL1-positive leukemia. Patients and Methods: Study subjects included 46 patients with newly diagnosed chronic myelogenous leukemia (CML), 24 with multidrug-resistant CML, 24 with BCR-ABL1-positive acute lymphocytic leukemia (ALL), as well as 38 healthy donors. Plasma cTK activity was measured using a chromogenic universal TK assay in which plasma samples and standards were added to wells of a microtiter plate that were coated with synthesized poly(Glu-Tyr) peptide substrates. Specific TK activity was measured by spectrophotometry, with or without addition of TKIs. Results: cTK activity was significantly higher in CML (median 801.93 U/mL, range 18.10–3932.30 U/mL) and BCR-ABL1-positive ALL patients (median 659.55 U/mL, range 0–1626.90 U/mL) than in healthy donors (median 82.85 U/mL, range 0.63–852.80 U/mL) (P<0.001). Plasma cTK activity was closely correlated with cellular BCR-ABL1 kinase activation, as indicated by phosphorylation of the downstream signaling proteins CRKL (P<0.001) and STAT-5 (P=0.003). However, cTK activity was independent of BCR-ABL1 transcript level and BCR-ABL1 mutation type. Plasma cTK activity was inhibited by imatinib and dasatinib in ex vivo studies; this inhibition was severely diminished in patients harboring T315I mutations. Conclusions: cTK activity is readily detectable in plasma or serum samples without pre-cleaning or enrichment. Ex vivo testing measuring the effect of TKIs on plasma cTK activity thus hold promise as drug sensitivity tests for predicting and monitoring response to specific TKIs. Disclosures: Yeh: Quest Diagnostics Inc.: Employment. Abdool:Quest Diagnostics Inc.: Employment. Bruey:Quest Diagnostics Inc.: Employment.

scholarly journals Ex Vivo Mitochondrial Respiration Parallels Biochemical Response to Ibrutinib in CLL Cells

Cancers ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 354
Author(s):  
Subir Roy Chowdhury ◽  
Cheryl Peltier ◽  
Sen Hou ◽  
Amandeep Singh ◽  
James B. Johnston ◽  
...  
Keyword(s):  
Mitochondrial Respiration ◽  
Ex Vivo ◽  
Standard Dose ◽  
Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Clinical Tool ◽  
Potential Biomarker ◽  
Apoptotic Pathways ◽  
The Impact

Mitochondrial respiration is becoming more commonly used as a preclinical tool and potential biomarker for chronic lymphocytic leukemia (CLL) and activated B-cell receptor (BCR) signaling. However, respiration parameters have not been evaluated with respect to dose of ibrutinib given in clinical practice or the effect of progression on ibrutinib treatment on respiration of CLL cells. We evaluated the impact of low and standard dose ibrutinib on CLL cells from patients treated in vivo on mitochondrial respiration using Oroboros oxygraph. Cytokines CCL3 and CCL4 were evaluated using the Mesoscale. Western blot analysis was used to evaluate the BCR and apoptotic pathways. We observed no difference in the mitochondrial respiration rates or levels of plasma chemokine (C-C motif) ligands 3 and 4 (CCL3/CCL4), β-2 microglobulin (β-2 M) and lactate dehydrogenase (LDH) between low and standard doses of ibrutinib. This may confirm why clinical observations of the safety and efficacy of low dose ibrutinib are observed in practice. Of interest, we also observed that the mitochondrial respiration of CLL cells paralleled the increase in β-2 M and LDH at progression. Our study further supports mitochondrial respiration as a biomarker for response and progression on ibrutinib in CLL cells and a valuable pre-clinical tool.

scholarly journals Semiautomated microtiter plate assay for monitoring peptidylprolyl cis/trans isomerase activity in normal and pathological human sera

1998 ◽  
Vol 44 (3) ◽  
pp. 502-508 ◽  
Author(s):  
Gerhard Küllertz ◽  
Sabine Lüthe ◽  
Gunter Fischer
Keyword(s):  
Biological Fluids ◽  
Microtiter Plate ◽  
Serum Samples ◽  
Ppiase Activity ◽  
Microtiter Plate Assay ◽  
Isomerase Activity ◽  
Microtiter Plates ◽  
Activity Factor ◽  
Healthy Donors ◽  
Human Sera

Abstract An UV/VIS spectrophotometric assay technique was developed that was able to routinely monitor peptidylprolyl cis/trans isomerase (PPIase) activity of biological fluids in 96-well microtiter plates. The assay, based on monitoring the cis-to-trans isomerization of succinyl-Phe-cisPro-Phe-4-nitroanilide as substrate in a chymotrypsin-coupled reaction, yields a throughput of 96 samples per 30 min. The assay’s capacity was exemplified by dealing with the PPIase activity in several normal and pathological human sera. Reference values of 151 healthy subjects (83 females, 69 males, 17 to 60 years old) were found to possess significant sex-specific differences. PPIase activity factor K of the sera was significantly greater in males (5th, 50th, 95th percentiles: 17, 36, 55 K) than females (14, 30, 48 K). PPIase activities of sera from healthy donors (n = 151) were significantly higher (Mann–Whitney rank-sum test P &lt;0.0001) than those of patients (n = 47). PPIase activity in serum samples stored at 4 °C was stable for at least 20 h.

scholarly journals Expression of axl, a transforming receptor tyrosine kinase, in normal and malignant hematopoiesis

Blood ◽  
1994 ◽  
Vol 84 (6) ◽  
pp. 1931-1941 ◽  
Author(s):  
A Neubauer ◽  
A Fiebeler ◽  
DK Graham ◽  
JP O'Bryan ◽  
CA Schmidt ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Receptor Tyrosine Kinase ◽  
Intracellular Signaling ◽  
Myeloid Leukemia ◽  
Blast Crisis ◽  
Chronic Phase ◽  
Lymphocytic Leukemia ◽  
Myelogenous Leukemia ◽  
Hematopoietic Tissue ◽  
Blood Leukocytes

Abstract We previously reported the cloning, and characterization of a receptor tyrosine kinase, axl, from two patients with chronic myelogenous leukemia. Herein, we describe the expression pattern of axl in normal and malignant hematopoietic tissue axl message is detected in normal human bone marrow but not significantly in normal blood leukocytes. Cell separation experiments showed that axl is expressed in hematopoietic CD34+ progenitor and marrow stromal cells, at low levels in peripheral monocytes, but not in lymphocytes or granulocytes. Consistent with the normal pattern of axl expression, axl RNA was found predominantly in diseases of the myeloid lineage: 39 of 66 (59%) patients with myeloproliferative disorders (acute myeloid leukemia, chronic myeloid leukemia (CML) in chronic phase, CML in myeloid blast crisis, and myelodysplasia) showed significant axl transcription, as compared with 1 of 45 (2%) lymphoid leukemias (chronic lymphocytic leukemia, acute lymphocytic leukemia, and CML in lymphoid blast crisis). Treatment of K562 cells with the phorbol ester, 12-O- tetradecanoylphorbol-13-acetate (TPA), administration of interferon alpha (IFN alpha) to normal monocytes, and treatment of U937 cells with TPA and IFN tau significantly induced axl expression, supporting a role for this kinase in the intracellular signaling of myeloid cells through a variety of biochemical pathways. These results suggest that the axl kinase may be operative in normal and malignant myeloid biology.

A Novel Mutation In Bruton Tyrosine Kinase Confers Acquired Resistance To Ibrutinib (PCI-32765) In CLL

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4914-4914 ◽  
Author(s):  
Richard R. Furman ◽  
Shuhua Cheng ◽  
Pin Lu ◽  
Menu Setty ◽  
Alexandar Perez ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Ex Vivo ◽  
Novel Mutation ◽  
Expression Profiles ◽  
Covalent Binding ◽  
Lymphocytic Leukemia ◽  
Brdu Incorporation ◽  
Bruton Tyrosine Kinase ◽  
Pre Treatment

Abstract The Bruton tyrosine kinase (BTK) inhibitor, ibrutinib has produced durable remissions in chronic lymphocytic leukemia (CLL). We describe a CLL patient who progressed while receiving ibrutinib following 20 months of once daily dosing. A cysteine-to-serine amino acid replacement was identified in BTK at position 481 that disrupts the covalent, but not non-covalent, binding of ibrutinib to BTK in silico structural modeling1. The mutation was present in relapsed samples while absent in the pre-treatment and responding samples. Following the mutation, the B cell receptor (BCR) pathway was reactivated as evidenced by increased cell signaling activities and gene expression profiles. Comparing the relapsed samples with the pre-treatment and responding samples, at the cellular level, mutated CLL cells displayed higher levels of the cell proliferation marker Ki67 in vivo and higher levels of ex-vivo BrdU incorporation. Transfection of the C481S mutant construct into a sensitive lymphoma cell line rendered it much more resistant to ibrutinib treatment demonstrating the cellular impact of the mutation (see attached graph). Interestingly, the ibrutinib-resistant CLL cells remained sensitive to other BCR inhibitors such as dasatinib and SYK inhibitors. These results confirm BTK as an important pharmacologic target of ibrutinib. Further, a mechanism of resistance was revealed, and alternative therapeutic options for ibrutinib resistance were explored. (First three authors with equal contribution) Disclosures: Furman: Genentech: Consultancy, Speakers Bureau; GlaxoSmithKline: Consultancy, Speakers Bureau; Pharmacyclics: Consultancy; Gilead: Consultancy. Chang:Pharmacyclics: Employment, Equity Ownership.

Phase I study of nilotinib in patients (pts) from Japan with imatinib-resistant Ph+ chronic myelogenous leukemia (CML) or acute lymphocytic leukemia (ALL)

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 17511-17511 ◽  
Author(s):  
A. Tojyo ◽  
Y. Miyazaki ◽  
N. Usui ◽  
Y. Kobyashi ◽  
S. Okamoto ◽  
...  
Keyword(s):  
Phase I ◽  
Phase I Study ◽  
Kinase Inhibitor ◽  
Systemic Exposure ◽  
Lymphocytic Leukemia ◽  
Myelogenous Leukemia ◽  
Serum Samples ◽  
Open Label ◽  
Dose Escalation Study ◽  
Imatinib Resistant

17511 Background: Nilotinib is a highly selective Bcr-Abl tyrosine kinase inhibitor designed to be more potent than imatinib. Results from a phase I study in Japanese pts with imatinib-resistant/intolerant Ph+CML/ALL are reported. Methods: Japanese pts with imatinib-resistant/intolerant Ph+CML (5 CP; 2 AP; 2 BC) or ALL (2) were enrolled in an open-label, dose-escalation study evaluating safety, efficacy and pharmacokinetics (PK) of oral nilotinib. A standard three-pt-per-cohort design was used; initial cohorts included 200mg QD, 400mg QD, and 400mg BID. Serum samples were collected on days 1 and 15 of cycle 1 in all 11 pts. Results: Peak serum concentration was achieved at 4 hrs in most pts. Systemic exposure increased with dose. Serum level reached steady-state within 1 week and was stable over time. The accumulation ratio (AUC day15/day1) was 2 to 3. Nilotinib 400mg BID maintained serum levels >/= 30 times higher than the IC50 required to inhibit Bcr-Abl phosphorylation. No pt experienced dose-limiting toxicity. 7/11 pts had Grade 3/4 AEs, only 1 pt had an AE (thrombocytopenia) suspected as being related to nilotinib treatment. No pt had EKG abnormalities. 57% of all pts and 75% of CML CP pts achieved MCyR. 3 pts in the 200mg QD cohort discontinued due to disease progression, all other pts completed the study. PK results are summarized in Table 1 . Conclusions: Nilotinib PK profile in Japanese pts is comparable to that reported for pts outside Japan. Based on these data, the recommended starting dose of nilotinib in Japanese pts with imatinib-resistant/intolerant Ph+ CML/ALL is 400mg BID. [Table: see text] No significant financial relationships to disclose.

Clinical pharmacokinetics (PK) of AMN107, a novel inhibitor of Bcr-Abl, in healthy subjects and patients with imatinib resistant or intolerant chronic myelogenous leukemia (CML) or relapsed/refractory Ph+ acute lymphocytic leukemia (Ph+ALL)

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 3095-3095 ◽  
Author(s):  
C. Tanaka ◽  
T. Smith ◽  
H. Kantarjian ◽  
F. Giles ◽  
O. Ottmann ◽  
...  
Keyword(s):  
Healthy Subjects ◽  
Systemic Exposure ◽  
Lymphocytic Leukemia ◽  
Myelogenous Leukemia ◽  
Time Data ◽  
Serum Samples ◽  
Clinical Pharmacokinetics ◽  
Abl Tyrosine Kinase ◽  
Imatinib Resistant ◽  
Effective Use

3095 Background: AMN107, a selective and potent inhibitor of Bcr-Abl tyrosine kinase, has demonstrated activity against imatinib resistant forms of Bcr-Abl in patients with CML or Ph+ALL in a phase I study. This study characterizes clinical PK of AMN107 for the safe and effective use of the drug. Methods: Serum samples were collected in 119 patients with CML or Ph+ALL treated with once (QD) or twice daily (BID) oral AMN107 (doses of 50 to 1200 mg/day). PK parameters were derived from serum concentration-time data by non-compartmental methods. In separate multi-period crossover studies 92 healthy subjects were treated with single doses of AMN107 to assess the food effect on PK, and to assess the potential for drug-drug interactions with medications which inhibit (ketoconazole) or are metabolized by CYP3A4 (midazolam). Results: Steady-state AMN107 PK in patients were stable over time, and showed a dose-dependency with less than dose-proportional increases in systemic exposure at dose levels higher than 400 mg QD. Daily exposure at 400 mg BID, which maintains serum AMN107 levels at least 30 times higher than the IC50 of cellular phosphorylation of Bcr-Abl, was greater than at 800 mg QD. The effective half-life was 15 hours. Bioavailability of AMN107 administered with a high-fat meal was increased by 82% compared to fasting. CYP3A4 inhibition by concomitantly administered ketoconazole produced 3-fold increases in systemic exposure to AMN107, whereas AMN107 appeared a weak inhibitor of CYP3A4 with 30% increase in systemic exposure of midazolam given concomitantly. Conclusions: Systemic AMN107 levels at an oral AMN107 dose of 400 mg BID in patients with CML or Ph+ALL were well above the IC50 of Bcr-Abl. The food/drug interaction results provide further guidance for the safe use of AMN107 in the clinical setting. [Table: see text]

Elevated serum ras level predicts decreased survival in patients with hematologic malignancies

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 6562-6562 ◽  
Author(s):  
E. M. Nelli ◽  
K. Leitzel ◽  
S. M. Ali ◽  
H. A. Al-Mondhiry ◽  
L. Demers ◽  
...  
Keyword(s):  
Overall Survival ◽  
Patient Group ◽  
Acute Myelogenous Leukemia ◽  
Elevated Serum ◽  
Hematologic Malignancies ◽  
Lymphocytic Leukemia ◽  
Myelogenous Leukemia ◽  
Ras Signaling ◽  
Potential Biomarker ◽  
Acute Myelogenous

6562 Background: Ras is a GDP/GTP binding G protein that acts as a molecular switch converting signals from the cell membrane to the nucleus to regulate cell proliferation, differentiation, and protein synthesis. Activation of ras oncogenes has been identified in a variety of cancers, including 30% of acute myelogenous leukemia patients. The purpose of our study was to evaluate serum ras levels and correlate with survival in hematologic cancer patients. Methods: A novel ras p21 ELISA (Oncogene Science/Bayer Diagnostics, Cambridge, MA) employing two monoclonal antibodies reactive with H, K, and N ras was utilized to quantify total ras levels in serum obtained from patients with various hematologic malignancies including acute myelogenous leukemia (AML), acute lymphocytic leukemia (ALL), chronic myelogenous leukemia (CML), and chronic lymphocytic leukemia (CLL). Results: The total leukemia patient group consisted of 52 patients. At the 75th percentile serum ras cutpoint (524 pg/ml) 11/52 patients were defined as elevated for serum ras. From this patient group, 38 patients had clinical followup available and were included in the Kaplan-Meier analysis of overall survival. Patients with elevated serum ras (>524 pg/ml) had significantly shorter overall survival compared to those without (median OS 205 vs. 677 days) (p= 0.04). In a multivariate analysis including serum ras level and type of leukemia, serum ras level remained a significant independent variable for shorter overall survival (p=0.004). Within leukemia subtypes 2/18 AML, 4/9 CML, 3/7 ALL, and 0/4 CLL patients had elevated serum ras levels. Conclusions: Leukemia patients with elevated serum ras levels had a significantly shorter overall survival. Serum ras should be evaluated as a potential biomarker in larger leukemia trials, especially for response to treatment with inhibitors of the ras signaling pathway. [Table: see text]

MEN1112/OBT357, an Anti Bst1/CD157 Humanized Antibody Inducing Acute Myelogenous Leukemia (AML) Blast Depletion in an Autologous Ex Vivo Assay: A Potential New Targeted Therapy for AML

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 788-788
Author(s):  
Adriano Venditti ◽  
Francesco Buccisano ◽  
Luca Maurillo ◽  
Maria Ilaria Del Principe ◽  
Andrea Coppola ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Peripheral Blood ◽  
Myeloid Leukemia ◽  
Ex Vivo ◽  
Myelogenous Leukemia ◽  
Monocytic Differentiation ◽  
Healthy Donors ◽  
Ex Vivo Assay ◽  
Acute Myeloid

Abstract Acute myeloid leukemia (AML) is a disease with a poor outcome and novel approaches are needed to improve survival and decrease toxicity of current therapies. Bst1/CD157 is a protein belonging to the ADP-ribosyl-cyclase family expressed on monocytes and neutrophils. This antigen was shown to be also expressed in peripheral blood (PB) and bone marrow (BM) blasts of acute myeloid leukemia (AML) patients either at primary diagnosis or at relapse(1,2,3). MEN1112/OBT357 is a humanized, de-fucosylated antibody targeting Bst1/CD157 with high affinity and developed to generate antibody dependent cell-mediated cytotoxicity (ADCC) response against AML blasts. Peripheral blood (PB) and bone marrow (BM) samples of 38 AML patients (29 at diagnosis, 6 at relapse, 3 resistant), have been analyzed for the expression of Bst1/CD157 on AML blast cells by fluorescence-activated cell sorting (FACS) using a PE conjugated form of MEN1112/OBT357. Bst1/CD157 expression has been confirmed in 91% and 96% of PB and BM AML samples, respectively. Furthermore, statistical analysis demonstrated that monocyte-oriented blasts are characterized by a brighter expression of Bst1/CD157 compared to blasts of non-monocytic lineage. The efficacy of MEN1112/OBT357 in depleting AML blasts was evaluated through FACS analysis in an autologous ex vivo assay performed on whole blood. The assay was set up using blood from healthy donors exposed to 10 μg/ml Rituximab for 18 hours to induce B cell depletion. In the same conditions, the ability of 10 μg/ml MEN1112/OBT357 to induce blasts depletion was tested.In whole PB,MEN1112/OBT357 was able to deplete AML blasts in 15/32 evaluable cases (46%). In BM, MEN1112/OBT357 induced blast depletion in 9/24 evaluable cases (36%). Interestingly, higher depletion rate was observed in relapse/refractory patients. When CD16A-158Phe/Val polymorphisms were analyzed utilizing a sequence based typing (SBT) assay, it was demonstrated that AML blast depletion was independent by FcRg polymorphism. Furthermore, no significant shedding of Bst1/CD157 antigen was observed in sera from AML patients, compared to the sera from patients with other hematologic diseases or healthy donors. In summary, we confirmed the frequent expression of Bst1/CD157 on blasts from AML patients, with the brightest pattern of positivity observed in cases belonging to monocytic differentiation lineage. MEN1112/OBT357 also induced a promising ADCC against AML blasts in an autologous setting, which is independent from FcR g phenotype. Since in vivo the exposure of AML blasts to MEN1112/OBT357 largely exceeds the incubation time of the depletion assay, we expect a further improvement of its anti-leukemic effect in the clinical setting. Based on these results, a phase I study in patients with relapsed or refractory AML has been initiated in December 2014. Disclosures Bellarosa: Menarini Ricerche: Employment. Bressan:Menarini Ricerche: Employment. Wilson:Oxford Biotherapeutics: Employment. Manzini:Menarini Ricerche: Employment. Capriati:Menarini Ricerche SpA: Employment. Simonelli:Menarini Ricerche SpA: Employment. Binaschi:Menarini Ricerche: Employment.

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