The influence of cytotoxic drugs on the immunophenotype of blast cells in paediatric B precursor acute lymphoblastic leukaemia

Antigens expressed on acute lymphoblastic leukaemia (ALL) blasts which are essential for planning the treatment or may influence the prognosis of the disease

Antigen	Expression of the antigen in normal and pathological conditions	Clinical significance
CD10	Normal lymphoid progenitors, neutrophils and blast cells of B-ALL¹⁹	Associated with favourable presenting features in paediatric B-ALL and possible target for emerging CAR-T^19,20,21
CD19	Normal and malignant B lymphocytes²²	Target of blinatumomab and CAR-T^22,23,24
CD20	Normal and malignant B lymphocytes²¹	Target of rituximab, up-modulated in B-ALL patients with poor prognosis^7,21
CD27	T cells, natural killer cells and thymocytes, also memory B cells, and in some subsets of B-ALL¹¹	Positive in B-ALL with BCR/ABL or CRLF2 rearrangement, high positivity was associated with poor prognosis^11,12,13
CD34	Pluripotent stem cells¹⁴	Lack of expression associated with worse event free and overall survival of ALL¹⁵
CD45	Cells of hematopoietic origin²⁵	High expression associated with high risk disease and worse event free survival, and worse rate of complete remission after the induction therapy^16,26
CD58	Hematopoietic and nonhematopoietic cells²⁷	Lack of expression reduces the efficacy of blinatumomab and anti-CD19 CAR-T^4,5
CD66c	Granulocytes and their precursors, most common myeloid antigen on malignant B lymphoblasts¹⁷	Associated with specific genetic alterations such as BCR/ABL, hypodiploidy, hypodiploidy and CRLF2-positivity in B-ALL^17,18
CD137	T lymphocytes and natural killer cells but also on activated B cells of naive origin²⁸	Enhances B cell survival²⁸

[i] B-ALL = B-cell ALL; CAR-T = chimeric antigen receptor T; CRLF2 = cytokine receptor-like factor 2

TABLE 2.

Clinical characteristics of all included patients

No. of all included patients with B-ALL	N = 29
Male/female	N
Male/female	17/12
Mean age at diagnosis	Years
Mean age at diagnosis	4.76 (newborn 17)
Flow cytometric findings at diagnosis	N (%)
Pro-B	3 (10)
Pre-B	6 (21)
Common type	20 (69)
Cytogenetic and molecular finding at diagnosis	N (%)
normal karyotype	6 (21)
hyperdiploid	10 (34)
t(12;21)	(28)
complex karyotype	(3)
MLL	1 (3)
changes of unknown risk potential	3 (10)
Outcome	N (%)
Alive	93)
Died	2 (7)
Cause of death	N
pancreatitis^*	1
progressive disease^**	1

* = treated according to ALL IC-BFM 2009 protocol;

** = treated according to Interfant 06 protocol; MLL = mixed-lineage leukaemia rearrangement; N = number of patients; Pro-B, Pre-B and common B = stage of differentiation

TABLE 3.

Response to the therapy in patients treated according ALL IC BFM 09 protocol

Patients treated according to ALL IC-BFM 2009 protocol
No. of patients	27
Treatment arm	N (%)
SR	6 (22)
IR	18 (66)
HR	3 (11)
Response to prednisolone on day 8	N (%)
good response^*	27 (100)
poor response^**	0 (0)
Minimal residual disease on day 15	N (%)
< 0.1%	10 (37)
0.1–10%	15 (56)
> 10%	2 (7)
Minimal residual disease on day 33	N (%)
neg	16 (59)
< 0.1%	6 (22)
0.1–10%	3 (11)
> 10%	2 (7)
Outcome	N (%)
alive	26 (96)
died	1 (4)

* = prednisolone good response is defined as blast count of less than 1000 per mL of peripheral blood on the day 8 of induction therapy;

** = prednisone poor response is defined as blast count of more than 1000 per mL of peripheral blood on the day 8 of induction therapy; HR = high risk; IR = intermediate risk; N = number of patients; SR = standard risk

TABLE 4.

The description of the antibodies used for flow cytometry

	Antibody	Antibody clone	Antibody vendor	Antibody volume (μL)
CD10	Anti-CD10 Horizon™ BV605	HI10a	BD Biosciences	5
CD19	CD19-BD Horizon™ V450 Mouse Anti-Human CD19	SJ25C1	BD Biosciences	5
CD20	CD20-BD™ CD20 APC	L27	BD Biosciences	7
CD27	CD27-BD Horizon™ APC-R700 Mouse Anti-Human CD27	M-T271	BD Biosciences	5
CD34	CD34-BD™ CD34 PE-Cy™7	8G12	BD Biosciences	5
CD45	CD45-BD™ CD45 APC-Cy™7	2D1	BD Biosciences	3
CD58	CD58-BD Pharmingen™ FITC Mouse Anti-Human CD58	1C3	BD Biosciences	3
CD66c	CD66c-BD OptiBuild™ BV510 Mouse Anti-Human CD66c	B6.2/CD66	BD Biosciences	5
CD137	CD137-BD Pharmingen™ PE Mouse Anti-Human CD137	4B4-1	BD Biosciences	7

Example of a gating procedure. Mononuclear cells were selected on the forward versus side scatter plot **(A)** and single cells by gating on the area against the width for forward scatter **(B)**. Propidium iodide (PI) was used to select viable cells **(C)**. The final selection of blasts was based on their immunophenotype, in this case by selecting CD10-positive cells on the CD10 versus CD45 plots **(D)**.

The plots of the marginal effects for the fixed effects coefficients of the models for CD137, CD34 and CD19. The lines correspond to the estimates of the ma rginal ef fects and the coloured areas to the 95% confidence intervals.

The plots of the marginal effects for the fixed effects coefficients of the models for CD20, CD45, CD27, CD66c, CD10, and CD58. The lines correspond to the estimates of the marginal effects and the coloured areas to the 95% confidence intervals.

TABLE 5.

The coefficients obtained by using separate mixed model which showed the effect of single drug in CDn antigen expression. The coefficient d showed the presence of CDn antigen and coefficient of k_d the influence of drug dose on antigen expression. The way how the given drug influences the antigen expression is shown in different colours: RED (coefficients d and k_d are statistically significantly different from 0), BLUE (the coefficient d is significantly different from 0), GREEN (coefficient k_d is significantly different from 0) and WHITE (neither the coefficient d nor k_d is statistically significantly different from 0). 95% confidence intervals, and corresponding p values are also shown. All confidence intervals and p values were adjusted with Holm’s method

Drug	CD	Coefficient d (presence)	p value for d	*Coefficient k_d* (log(amount))**	p value for k_d
Asparaginase	CD10	−0.12, [−0.33, 0.1]	1	−0.01, [−0.11, 0.1]	1
	CD137	−0.18, [−0.46, 0.1]	1	0, [−0.11, 0.12]	1
	CD19	−0.09, [−0.42, 0.24]	1	−0.01, [−0.14, 0.13]	1
	CD20	−0.15, [−0.39, 0.09]	1	0, [−0.09, 0.1]	1
	CD27	−0.03, [−0.28, 0.22]	1	−0.01, [−0.12, 0.11]	1
	CD34	−0.15, [−0.4, 0.09]	1	−0.09, [−0.23, 0.04]	1
	CD45	−0.04, [−0.27, 0.2]	1	−0.02, [−0.16, 0.11]	1
	CD58	−0.05, [−0.23, 0.13]	1	0.01, [−0.08, 0.1]	1
	CD66c	−0.05, [−0.36, 0.27]	1	0.02, [−0.15, 0.19]	1
Daunorubicin	CD10	−1.1, [−1.38, −0.83]	< 0.001	−0.47, [−0.63, −0.31]	< 0.001
	CD137	1.05, [0.72, 1.37]	< 0.001	0.4, [0.21, 0.59]	< 0.001
	CD19	−1.05, [−1.48, −0.62]	< 0.001	−0.35, [−0.6, −0.1]	< 0.001
	CD20	−0.24, [−0.53, 0.05]	0.35	−0.22, [−0.39, −0.05]	0.002
	CD27	−0.6, [−0.92, −0.27]	< 0.001	−0.09, [−0.27, 0.09]	1
	CD34	−1.48, [−1.8, −1.16]	< 0.001	−0.74, [−0.93, −0.55]	< 0.001
	CD45	−0.58, [−0.9, −0.27]	< 0.001	−0.28, [−0.47, −0.1]	< 0.001
	CD58	−0.73, [−0.97, −0.5]	< 0.001	−0.32, [−0.46, −0.19]	< 0.001
	CD66c	0.28, [−0.13, 0.69]	1	0.13, [−0.1, 0.37]	1
Methotrexate	CD10	−0.1, [−0.52, 0.33]	1	−0.04, [−0.32, 0.24]	1
	CD137	0.11, [−0.33, 0.56]	1	0.02, [−0.24, 0.29]	1
	CD19	−0.33, [−1, 0.35]	1	0.02, [−0.38, 0.42]	1
	CD20	−0.21, [−0.68, 0.27]	1	0.04, [−0.26, 0.34]	1
	CD27	−0.26, [−0.79, 0.27]	1	−0.03, [−0.34, 0.29]	1
	CD34	0.08, [−0.4, 0.56]	1	0.04, [−0.28, 0.35]	1
	CD45	−0.04, [−0.5, 0.41]	1	0.03, [−0.29, 0.35]	1
	CD58	0.01, [−0.25, 0.27]	1	0.02, [−0.21, 0.26]	1
	CD66c	−0.05, [−0.64, 0.54]	1	−0.02, [−0.4, 0.37]	1
Prednisolone	CD10	−0.42, [−0.64, −0.21]	< 0.001	−0.01, [−0.12, 0.1]	1
	CD137	−0.06, [−0.32, 0.19]	1	0.05, [−0.1, 0.19]	1
	CD19	−0.61, [−0.94, −0.27]	< 0.001	−0.03, [−0.2, 0.15]	1
	CD20	0.14, [−0.09, 0.37]	1	0.02, [−0.1, 0.14]	1
	CD27	−0.5, [−0.76, −0.23]	< 0.001	−0.15, [−0.3, 0]	0.055
	CD34	−0.35, [−0.6, −0.11]	< 0.001	0.03, [−0.1, 0.16]	1
	CD45	−0.12, [−0.36, 0.12]	1	−0.07, [−0.2, 0.07]	1
	CD58	−0.19, [−0.38, −0.01]	0.034	−0.05, [−0.15, 0.05]	1
	CD66c	0, [−0.19, 0.19]	1	−0.02, [−0.2, 0.15]	1
Vincristine	CD10	−0.14, [−0.37, 0.1]	1	−0.05, [−0.14, 0.05]	1
	CD137	0.01, [−0.22, 0.25]	1	0.07, [−0.04, 0.18]	1
	CD19	−0.56, [−0.94, 0]	< 0.001	−0.17, [−0.32, −0.03]	0.004
	CD20	0.06, [−0.19, 0.31]	1	0.01, [−0.08, 0.09]	1
	CD27	−0.06, [−0.34, 0.23]	1	0, [−0.09, 0.09]	1
	CD34	−0.18, [−0.45, 0.1]	1	−0.04, [−0.14, 0.06]	1
	CD45	0.36, [0.07, 0.64]	0.002	0.16, [0.05, 0.26]	< 0.001
	D58	−0.27, [−0.48, −0.06]	0.001	−0.11, [−0.19, −0.03]	< 0.001
	CD66c	−0.04, [−0.39, 0.3]	1	−0.02, [−0.15, 0.11]	1

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DOI: https://doi.org/10.2478/raon-2024-0006 | Journal eISSN: 1581-3207 | Journal ISSN: 1318-2099

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Language: English

Page range: 133 - 144

Submitted on: May 14, 2023

Accepted on: Dec 6, 2023

Published on: Feb 21, 2024

Published by: Association of Radiology and Oncology

In partnership with: Paradigm Publishing Services

Publication frequency: 4 issues per year

Keywords:

immunophenotypic changes,

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© 2024 Tomaz Prelog, Simon Bucek, Andreja Brozic, Jakob Peterlin, Marko Kavcic, Masa Omerzel, Bostjan Markelc, Tanja Jesenko, Veronika Kloboves Prevodnik, published by Association of Radiology and Oncology
This work is licensed under the Creative Commons Attribution 4.0 License.

Volume 58 (2024): Issue 1 (March 2024)

The influence of cytotoxic drugs on the immunophenotype of blast cells in paediatric B precursor acute lymphoblastic leukaemia

Figures & Tables

TABLE 1.

TABLE 2.

TABLE 3.

TABLE 4.

FIGURE 1.

FIGURE 2A.

FIGURE 2B.

TABLE 5.

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