IMR Press / FBL / Volume 27 / Issue 2 / DOI: 10.31083/j.fbl2702039
Open Access Original Research
Cytofluorimetric assay to investigate variability in blinatumomab in vitro response
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1 Department of Medical, Surgical and Health Sciences, University of Trieste, 34149 Trieste, Italy
2 Azienda Sanitaria Universitaria Giuliano Isontina (ASUGI), 34148 Trieste, Italy
3 Institute for Maternal & Child Health (I.R.C.C.S) Burlo Garofolo, 34137 Trieste, Italy
4 Department of Life Sciences, University of Trieste, 34127 Trieste, Italy

Academic Editors: Yingqun Wang and Haihua Feng

Front. Biosci. (Landmark Ed) 2022, 27(2), 39;
Submitted: 25 October 2021 | Revised: 19 December 2021 | Accepted: 20 December 2021 | Published: 24 January 2022
(This article belongs to the Special Issue Novel Approaches to Cancer Diagnosis and Therapy)
Copyright: © 2022 The Author(s). Published by IMR Press.

This is an open access article under the CC BY 4.0 license.


Background: The T-cell engager antibody blinatumomab (BlincytoTM) represents a promising rescue therapy for relapsed/refractory CD19+ acute lymphoblastic leukemia (B-ALL), although ~20–30% of patients still do not respond to treatment. Blinatumomab creates a tight synapsis between CD3+ T-lymphocytes and leukemic CD19+ B-cells, resulting in a granzyme B (GzB)-mediated specific lysis of leukemic cells. Methods: Aim of the study was to provide evidence that variability in blinatumomab response could have a genetic basis in PAX5, one of the most often mutated genes in B-ALL, affecting the CD19 surface expression on lymphoblasts, and could be explored in vitro by means of a cytofluorimetric assay, staining both surface antigens (CD45, CD19 and CD3) and intracytoplasmic markers (7AAD, Syto16). Two human immortalized B-ALL cell lines (NALM6 and REH) were chosen for their different PAX5 and CD19 protein levels, as verified by western blot and flow cytometry, respectively. Results: In contrast to NALM6, REH cells do not express the full-length PAX5 protein and show less CD19 on the cell surface (fluorescence peak median intensity: 9155 versus 28895). Co-cultures of CD3+ T-lymphocytes from healthy donors and B-ALL cell lines were seeded at an effector-to-target cell ratio of 1:10 for simulating the condition existing in the bone marrow due to the malignant invasion of blast cells. Co-cultures were exposed in vitro to blinatumomab and the simultaneous increase in blast mortality and T-lymphocytes activation induced by the drug was observed at day +7 (both effects: p < 0.0001 versus untreated, two-way ANOVA, Bonferroni post-test), and was particularly pronounced in REH compared to NALM6 co-cultures (p < 0.05). Surprisingly, daily release of GzB in supernatants, measured by an ELISA assay, was significantly lower in drug-exposed REH co-cultures compared to NALM6 at early time-points (days +3 and +4, p-value < 0.0001, three-way ANOVA), reaching a comparable plateau only towards the end of the incubation period (at day +5). Only 2 out of 5 primary co-cultures of leukemic and mononuclear cells isolated from bone marrow aspirates of B-ALL patients (age: median 10.7 years, interquartile range (IQR) 3.4; males: 60%) responded to the drug in vitro (simultaneous blast mortality and T-lymphocyte activation: both effects: p < 0.0001 versus untreated) and at different drug concentrations. Results were unrelated to the percentages of immature CD19+ B-cells in the diagnostic samples. Conclusions: In conclusion, cytofluorimetric analysis can highlight the different response induced by blinatumomab among co-cultures. Whether and how this difference is affected by PAX5-regulated CD19 expression is unclear and whether it is predictive of in vivo response to therapy remains to be established. Further dedicated studies are required to investigate these issues in detail.

Pediatric acute lymphoblastic leukemia
In vitro cytofluorimetric assay
Fig. 1.
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