5-5--6-6--tetrachloro-1-1--3-3--tetraethylbenzimidazolocarbocyanine and Leukemia--Myeloid--Acute

Chemotherapy resistance is the main cause of treatment failure in acute myeloid leukemia (AML) and has been related to ATP-binding cassette (ABC) transporter activity. However, the links between ABC activity, immunophenotype, and molecular AML parameters have been poorly evaluated. Moreover, the prognostic value of ABC activity, when compared to new molecular markers, is unknown. Here we investigated the links between ABC activity, as evaluated by JC-1 +/- cyclosporine A assay, and immunophenotypic, cytogenetic, molecular, and targeted next-generation sequencing features in 361 AML patients. High ABC activity was found in 164 patients and was significantly associated with less proliferating disease, an immature immunophenotype (expression of CD34, HLA-DR, CD117, CD13), and gene mutations defining AML as belonging to secondary-type ontogenic groups. Low ABC activity was associated with more mature myeloid differentiation (CD34-, cyMPO+, CD15+, CD33+) or monocytic commitment (CD64+, CD4+weak, CD14+), with NPM1 mutations, KMT2A rearrangements, and core-binding factor gene fusions, hallmarks of the de novo-type AML ontogeny. ABC activity was one of the major factors we identified using a random forest model for early prediction of AML ontogeny. In the 230 patients evaluated at diagnosis and intensively treated, high ABC activity was a predictive factor for primary resistance, and in multivariate analysis including full molecular data, an independent factor for event-free survival (P=0.0370). JC-1 +/- cyclosporine A assay could be used at diagnosis to predict AML ontogeny and to complete prognosis evaluation in addition to new molecular markers.

Topics: Adult; Antigens, CD34; Cyclosporine; HLA-DR Antigens; Humans; Immunophenotyping; Leukemia, Myeloid, Acute; Prognosis

The purpose of the study is to establish a simple and relatively inexpensive flow cytometric chemosensitivity assay (FCCA) for leukemia to distinguish leukemic blasts from normal leukocytes in clinical samples.. We first examined whether the FCCA with the mitochondrial membrane depolarization sensor, 5, 50, 6, 60-tetrachloro-1, 10, 3, 30 tetraethyl benzimidazolo carbocyanine iodide (JC-1), could detect drug-induced apoptosis as the conventional FCCA by annexin V/7-AAD detection did and whether it was applicable in the clinical samples. Second, we compared the results of the FCCA for prednisolone (PSL) with clinical PSL response in 18 acute lymphoblastic leukemia (ALL) patients to evaluate the reliability of the JC-1 FCCA. Finally, we performed the JC-1 FCCA for bortezomib (Bor) in 25 ALL or 11 acute myeloid leukemia (AML) samples as the example of the clinical application of the FCCA.. In ALL cells, the results of the JC-1 FCCA for nine anticancer drugs were well correlated with those of the conventional FCCA using anti-annexin V antibody (P < 0.001). In the clinical samples from 18 children with ALL, the results of the JC-1 FCCA for PSL were significantly correlated with the clinical PSL response (P = 0.005). In ALL samples, the sensitivity for Bor was found to be significantly correlated with the sensitivity for PSL (P = 0.005). In AML samples, the Bor sensitivity was strongly correlated with the cytarabine sensitivity (P = 0.0003).. This study showed the reliability of a relatively simple and the FCCA using JC-1, and the possibility for the further clinical application.

Topics: Adolescent; Annexin A5; Antineoplastic Agents; Apoptosis; Benzimidazoles; Carbocyanines; Cell Line, Tumor; Child; Child, Preschool; Flow Cytometry; Fluorescent Dyes; Humans; Infant; Leukemia, Myeloid, Acute; Membrane Potential, Mitochondrial; Precursor B-Cell Lymphoblastic Leukemia-Lymphoma; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Reproducibility of Results; Sensitivity and Specificity

We studied expression and functional activity of tumor cell P-gp in children with various leukemia variants and analyzed its prognostic role in acute lymphoblastic leukemia. Functional activity of P-gp increased in acute myeloid leukemia, relapses of acute lymphoblastic leukemia (in comparison with primary disease), and in a group of patients in whom no remission was attained. The survival of patients with acute lymphoblastic leukemia was lower in cases with increased expression and function of P-gp.

Topics: Adolescent; Antibodies, Monoclonal; ATP Binding Cassette Transporter, Subfamily B, Member 1; Benzimidazoles; Carbocyanines; Child; Child, Preschool; Flow Cytometry; Gene Expression; Humans; Infant; Leukemia, Myeloid, Acute; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Prognosis; Statistics, Nonparametric; Survival Analysis

One of the possible causes of treatment failure in acute leukemia is the emergence of multidrug resistance caused by P-glycoprotein (P-gp) overexpression. We compared a flow cytometric assay using JC-1 with a technique using rhodamine 123 (rho123) to evaluate the P-gp function in acute leukemia. Samples from 50 acute leukemia patients were analyzed by both functional assays. The P-gp expression was assessed by an immunological flow cytometric test and the association between the P-gp status and the clinical outcome was evaluated. Of all samples, 28% showed a reversible JC-1 efflux and 36% scored positive for the rho123 assay. In two cases, the leukemic blasts showed a reversible JC-1 efflux whereas they were negative for rho123. These patients had blast cells with a very low P-gp activity. Six samples scored positive for the rho123 assay but were negative for the JC-1 test. Five of these samples did not express P-glycoprotein and were considered false positive. We found a strong correlation between the JC-1 and the rho123 test (R(s)=0.59, p<0.0001) and the JC-1 and the immunological assay (R(s)=0.29, P=0.05). There was also an association between the JC-1 status and the clinical outcome of adult patients (chi2=6.30, P=0.04). In conclusion, we recommend the JC-1 assay to study the P-gp activity in acute leukemia because it is more specific and less labor intensive than conventional functional flow cytometric tests using rhodamine 123. In addition, the JC-1 assay can be used to identify adult patients with an increased risk for adverse clinical outcome.

Topics: Adult; ATP Binding Cassette Transporter, Subfamily B, Member 1; Benzimidazoles; Carbocyanines; Child; Drug Resistance, Neoplasm; Female; Flow Cytometry; Fluorescent Dyes; Humans; K562 Cells; Leukemia; Leukemia, Myeloid, Acute; Male; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Rhodamine 123; Risk; Treatment Outcome