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

Tomaz Prelog

Tomaz Prelog

  • Department of Haemato-Oncology, University Children’s Hospital, University Medical Centre LjubljanaLjubljana, Slovenia
  • Faculty of Medicine, University of LjubljanaLjubljana, Slovenia
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Prelog, Tomaz
, 
Simon Bucek

Simon Bucek

Department of Cytopathology, Institute of Oncology LjubljanaLjubljana, Slovenia
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Bucek, Simon
, 
Andreja Brozic

Andreja Brozic

Department of Cytopathology, Institute of Oncology LjubljanaLjubljana, Slovenia
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Brozic, Andreja
, 
Jakob Peterlin

Jakob Peterlin

Institute for Biostatistics and Medical Informatics, University of LjubljanaLjubljana, Slovenia
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Peterlin, Jakob
, 
Marko Kavcic

Marko Kavcic

Department of Haemato-Oncology, University Children’s Hospital, University Medical Centre LjubljanaLjubljana, Slovenia
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Kavcic, Marko
, 
Masa Omerzel

Masa Omerzel

Department of Experimental Oncology, Institute of Oncology LjubljanaLjubljana, Slovenia
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Omerzel, Masa
, 
Bostjan Markelc

Bostjan Markelc

Department of Experimental Oncology, Institute of Oncology LjubljanaLjubljana, Slovenia
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Markelc, Bostjan
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Tanja Jesenko

Tanja Jesenko

  • Faculty of Medicine, University of LjubljanaLjubljana, Slovenia
  • Department of Experimental Oncology, Institute of Oncology LjubljanaLjubljana, Slovenia
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Jesenko, Tanja
 and 
Veronika Kloboves Prevodnik

Veronika Kloboves Prevodnik

  • Faculty of Medicine, University of LjubljanaLjubljana, Slovenia
  • Department of Cytopathology, Institute of Oncology LjubljanaLjubljana, Slovenia
  • Institute of Pathology, Faculty of Medicine, University of MariborMaribor, Slovenia
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Prevodnik, Veronika Kloboves
  
Feb 21, 2024
The influence of cytotoxic drugs on the immunophenotype of blast cells in paediatric B precursor acute lymphoblastic leukaemia's Cover Image
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Article Category: Research Article
Published Online: Feb 21, 2024
Page range: 133 - 144
Received: May 14, 2023
Accepted: Dec 06, 2023
DOI: https://doi.org/10.2478/raon-2024-0006
Keywords
© 2024 Tomaz Prelog et al., published by Sciendo
This work is licensed under the Creative Commons Attribution 4.0 International License.

FIGURE 1.

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).
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).

FIGURE 2A.

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 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.

FIGURE 2B.

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.
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.

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)

Clinical characteristics of all included patients

No. of all included patients with B-ALL N = 29
Male/female N
17/12
Mean age at diagnosis Years
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

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

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-T19,20,21
CD19 Normal and malignant B lymphocytes22 Target of blinatumomab and CAR-T22,23,24
CD20 Normal and malignant B lymphocytes21 Target of rituximab, up-modulated in B-ALL patients with poor prognosis7,21
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 prognosis11,12,13
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 therapy16,26
CD58 Hematopoietic and nonhematopoietic cells27 Lack of expression reduces the efficacy of blinatumomab and anti-CD19 CAR-T4,5
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-ALL17,18
CD137 T lymphocytes and natural killer cells but also on activated B cells of naive origin28 Enhances B cell survival28

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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