Flow cytometry is one of the methods used in the diagnosis of acute lymphoblastic leukaemia (ALL). It provides important information about the expression of antigens on blast cells and is important for determining minimal residual disease (MRD), which allows assessment of response to therapy and further planning of treatment.1,2
According to the International Berlin-Frankfurt-Münster Study Group clinical trial (ALL IC BFM 2009) the intensity of therapy for childhood ALL is based on risk stratification.1 The patients are divided into three risk groups, standard risk (SR), intermediate risk (IR) and high risk (HR). HR patient must fulfil at least one of the following criteria: presence of specific genetic alterations (t(9;22) or t(4;11)), hypodiploid blast cells, > 1000 blast cells in 1 mL peripheral blood on day 8 of therapy, MRD on day 15 > 10% and patients not in morphological remission on day 33.1 SR patients are 1–6 years old, have a white blood cell count of < 20000/mL at the time of diagnosis, < 1000 blasts per mL of peripheral blood on day 8 of therapy, MRD on day 15 < 0.1% and are in morphological remission on day 33 of therapy.1 All others are IR patients.1
SR and IR patients are treated with 4 consecutive chemotherapy blocks: induction, early intensification, consolidation and reinduction, followed by maintenance therapy. HR patients receive more intensive consolidation with 6 blocks of chemotherapy or 3 blocks followed by bone marrow transplantation.1 In induction prednisolone, vincristine, daunorubicin, asparaginase and intrathecal application of methotrexate are used.1 In later phases, other cytostatic drugs are administered.1
In relapsed or refractory disease, when immunotherapy with bispecific antibodies or chimeric antigen receptor T (CAR-T) cells is commonly indicated, the choice of therapeutic approach is often based on the immunophenotype of the blast cells. For instance the expression of antigens such as CD19, CD20 and CD58 is essential for planning treatment with blinatumomab, rituximab and CAR-T therapy.2,3,4,5,6
Some studies have already investigated the impact of the cytotoxic drugs on antigen expression of blast cells and showed that chemotherapy can cause a change in their expression, which may be related to cell death, the efficacy of therapy and the overall prognosis of the disease.7,8,9,10 Moreover, chemotherapy may also influence expression of antigens which are essential for effective treatment with bispecific antibodies and CAR-T cells.10
Certain antigens expressed on blast cells may also be associated with the prognosis of the ALL patients. Expression of CD27, CD34, CD45 and CD66c antigens has already been reported to be associated with prognosis of ALL.11,12,13,14,15,16,17,18 Additionally, expression of CD66c strongly correlates with BCR/ABL rearrangement.17 To our knowledge there is no data about the influence of chemotherapeutic drugs on expression of these prognostic markers in the literature. In Table 1 we present the antigens which are important for planning the treatment of ALL patients or are associated with the prognosis of the ALL.
Antigens expressed on acute lymphoblastic leukaemia (ALL) blasts which are essential for planning the treatment or may influence the prognosis of the disease
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 |
B-ALL = B-cell ALL; CAR-T = chimeric antigen receptor T; CRLF2 = cytokine receptor-like factor 2
The aim of the study was to assess the influence of drugs included in ALL induction therapy on antigen expression of blast cells which are common targets for immunotherapy (CD19, CD20) or possess the potential to impact its effectiveness (CD58). Additionally, we have studied the antigens that could play a role in categorizing patients into distinct treatment risk groups, enhancing the precision of patient stratification (CD10, CD27, CD34, CD45, CD66c and CD137). Importantly, we analysed the influence of drugs and their dosage separately.
Between March 2018 and November 2021, we performed bone marrow aspiration or peripheral blood withdrawal in case of extreme leukocytosis in all children with suspected acute leukaemia. Informed consent was obtained from the patient and parents or consent from a proxy before enrolment in the study. The National Medical Ethics Committee approved the study (protocol number: KME 25/05/15).
First, we performed all routine diagnostic procedures, and if B-cell ALL was confirmed, the remaining bone marrow sample was further cultivated
Twenty-seven of the included patients older than one year were treated according to the ALL IC-BMF 09 protocol1, more than half of them according to the intermediate risk arm. Two patients younger than 1 year were treated according to the Interfant 06 protocol.29 None of the included patients had blasts in the cerebrospinal fluid at the time of diagnosis. One patient who failed induction therapy received anti-CD19 CAR-T cell therapy, followed by bone marrow transplantation. Two other high-risk patients were treated with allogeneic stem cell transplantation. The clinical characteristics and outcome of all included patients are summarised in Table 2.
Clinical characteristics of all included patients
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 |
= 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
The response to therapy in patients treated according to the ALL IC-BFM 09 protocol is summarised in Table 3.
Response to the therapy in patients treated according ALL IC BFM 09 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
Among two patients, who were treated according to Interfant 06 protocol one of them had extreme hyperleukocytosis, finished the therapy and is in a remission. The other patient died of progressive disease.
After confirming B-ALL diagnosis, blast cells from bone marrow or peripheral blood were isolated by density gradient centrifugation using Ficoll-Paque (GE HealthCare Technologies Inc, Chicago, IL, USA) media and SepMateTM-15 centrifugation tubes (STEMCEL Technologies, Vancouver, Canada). After centrifugation and harvesting from the top of the Ficoll layer, cells were washed 2 times in RPMI 1640 medium containing 10 mM HEPES buffer (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). Medium was supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco), Gluta-MAX (100 ×, Gibco), and penicillin-streptomycin (100 ×, Sigma-Aldrich, Merck, Darm-stadt, Germany). Cells were plated at high density in T-75 or T-182 flasks (VWR, Radnor, PA, USA) in cell culture media and incubated in a humidified incubator with 5% CO2 at 37°C until the treatment with cytotoxic drugs (maximum 3 days).
Cells were collected and a suspension of 1×106 cells in 1.8 mL of fresh culture medium was plated in each well of the 24-well plate (Corning Inc., Corning, NY, USA). Then, 0.2 mL of different concentrations of cytotoxic drugs (prednisolone, daunorubicin, methotrexate, asparaginase or vincristine) diluted in saline were added to the cells. Each drug was used in three logarithmically increasing concentrations, which were determined before the beginning of the study. Few logarithmically increasing concentrations were tested and final concentrations used in the study were selected based on blast viability. More specifically, the lowest concentration was chosen such that it was not significantly cytotoxic. In the experiments, prednisolone (Predisolut, MIBE GmbH Arzneimittel, Brehna, Germany) was used at final concentrations of 1, 0.1 and 0.01 mM, daunorubicin (Daunoblastin, Pfizer, New York, NY, USA) at concentrations of 1, 0.1 and 0.01 µg/mL, methotrexate (Methotrexate, Medac Pharma, Wedel, Germany) at concentrations of 1, 0.1 and 0.01 mg/mL and asparaginase (Oncaspar Pegaspargase, Servier Pharmaceuticals LLC, Boston, MA; USA) at concentrations of 1, 0.1 and 0.01 units/mL. Vincristine (Vicristine, Teva Pharmaceuticals, Tel Aviv, Israel) was used at concentrations of 0.1, 0.01 and 0.001 µg/mL, with the exception of two experiments, performed at the time when optimal concentration was to be determined. In those two experiments vincristine was used at concentration of 10, 1 and 0,1 µg/mL. Each experiment also included a population of blast cells not exposed to any cytotoxic drug (addition of 0.2 mL saline), which was used as a control. After the addition of the cytotoxic drugs, the cells were incubated in a 5% CO2 humidified incubator at 37 °C for three days and then prepared for immunophenotypic analysis by quantitative flow cytometry.
To quantify the antigen expression a calibration chart was used. It was based on Quantum™ Simply Cellular® beads (Bangs Laboratories Inc., Fishers, IN, USA) and was generated at the time of each experiment. The sample and bead measurements were performed on the same day under the same conditions. The measured fluorescence of each antibody on the surface of the blast cells was converted to an antibody count using the calibration chart and expressed as antibody binding capacity. Measurements, analysis and interpretation of the results were performed according to the Instructions for Use of Quantum™ Simply Cellular® Beads (Bangs Laboratories Inc.).
Cell count and sample preparation for flow-cytometric analysis was carried out as previously described by our group.30,31 The sample aliquot containing half a million cells was first put in a test tube. Antibodies against CD10, CD19, CD20, CD27, CD34, CD45 CD58, CD66c and CD137 antigens were added at saturation concentrations (Table 4). 1µL of propidium iodide (concentration 200µg/mL) was also added to determine the viability of cells. After 20 min incubation, erythrocyte lysis was carried out using a commercial lysing solution (BD Biosciences, Franklin Lakes, NJ, USA). The samples were than acquired using a FACSCanto 10-colour flow cytometer (BD Biosciences) with three lasers (405, 488 and 633 nm) and FACSDiva 8.0.2 software (BD Bioscience). The flow cytometer was routinely set up and calibrated by measuring FACSDivaTM CS &T IVD beads (BD Biosciences) and SpheroTM Rainbow Calibration particles (Spherotech Inc., Lake Forest, IL, USA).
The description of the antibodies used for flow cytometry
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 |
For the final analyses, the software BD FlowJo 10.8.1 (BD Biosciences) was used. First a gate for mononuclear cells on the forward versus side scatter plot was selected. In the next step, single cells were selected by gating on the height against the area for forward scatter. Gating on propidium iodide was used to select only viable cells. In the final step, blast cells were gated according to their immunophenotype, in most cases by selecting CD10-positive cells on CD10 versus CD45 plots. An example of a gating procedure is shown in Figure 1. Finally, we determined the fluorescence of each antigen analysed from the histogram.
The analysis was performed using R (R version 4.1.0)32 and Julia (version 19.2).33 Linear mixed models were fitted using R package nlme (version 3.1–157)34 and using MixedModels.jl (version 4.13.1)35 in Julia. The optimal transformation of the dependent variables and the assessment of the goodness of fit of linear mixed models were performed with the help of the goodness of fit procedure described in publication by Peterlin
We have analyzed every single antigen expression (CDn) with a separate linear mixed model. Before analyzing the data with linear mixed models, we transformed the dependent variables CDn and the number of cytotoxic drugs given. We have done this to improve the resulting models’ goodness of fit. We have used 9 models described by the formula:
In the above formula, the term
For every drug In the case of CD19 and vincristine, we can therefore expect that the value of the log( In the case of prednisolone and CD19, we can say that we expect that the patient’s value of log( Other combinations of drug and observed antigen with the same pattern of influence were daunorubicin and CD27, as well as prednisolone and CD10, CD27, CD34 and CD58. In the case of daunorubicin and CD20, we can say that the value of patients’ log( Marginal effects for these nine models are shown in Figure 2A and Figure 2B. From these figures, we can easily see which cytotoxic drugs change a specific CDn antigen expression. However, since this study is primarily concerned with the effects of cytotoxic drugs conditionally on the individual and these figures are primarily informative, the confidence intervals in these two figures are not adjusted.
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:
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 | |
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 | |
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 | |
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 | |
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 |
Our
CD20 expression was stable with all cytotoxic drugs except daunorubicin. When the effect of daunorubicin was analysed with our statistical model, there were no changes in CD20 expression. However, when only the role of dose was examined, a modulation of CD20 expression was observed. Accordingly, we cannot say that just giving some amount of daunorubicin to blast cells causes an up- or down-modulation of CD20 expression without specifying the amount of the drug. These results are difficult to interpret, but as our study was not designed to explain the isolated effect of the dose when there is no effect of the drug in the model. To clarify the discrepancy between the effect of the drug and its dose, further analyses with a larger number of patients and a larger number of different concentrations of daunorubicin are needed.
Dworzak
Rituximab, an anti-CD20 antibody, has been shown to improve survival in adult patients with CD20-positive B-cell ALL.2 Based on the data published by van der Sluijs-Gelling
CD19 expression in ALL is of clinical importance due to the efficacy of anti-CD19 therapies such as blinatumomab or CAR-T cells.23,24 According to our results, daunorubicin, vincristine and prednisolone can induce down-modulation of CD19, which is partly consistent with data published by Van der Sluijs-Gelling
The presence of CD58 has been shown to be very important for the efficacy of bispecific anti-CD19 antibodies and anti-CD19 CAR-T cell therapy, as the lack of CD58 decreases T-lymphocyte activation and reduces the success of treatment.4,5 In our experiment, CD58 was down-modulated when daunorubicin, prednisolone or vincristine was added. The effect of prednisolone was dose-independent, while the dose of daunorubicin and vincristine was also important. Gaipa
Specific changes in CD10 and CD34 expression during ALL therapy is controversial, but it has been shown that CD10 positivity in B-ALL had been associated with disease prognosis.15 CD10, also known as the common acute lymphoblastic leukaemia antigen, can be detected in the blast cells of most patients with B-ALL.15 The prognostic significance of CD10 expression is associated with several favourable features such as a hyperdiploid karyotype, age less than nine years and a standard risk group.15,19 Our study showed down-modulation of CD10 after treatment with daunorubicin or prednisolone, and the effect of prednisolone was dose-independent. This is consistent with published data showing a transient down-modulation of CD10 in the early phase of ALL treatment, with the changes being more pronounced in patients with a good response to prednisolone and a better prognosis of the disease.8,9,10,37
CD34, the pluripotent stem cell antigen, plays an important role in stem cell attachment to the extra-cellular matrix and its presence on blast cells has been shown to be a good prognostic factor in adult patients with ALL, who have undergone stem cell transplantation in the first remission.14 Its positivity in ALL is also associated with a lower leukocyte count, a favourable karyotype and a better prognosis compared to CD34-negative ALL.15 In our experiment, prednisolone and daunorubicin triggered a significant down-modulation of CD34, which again is partly consistent with published data, where down-modulation of CD34 on blast cells after
In our study CD10 and CD34 were shown to be down-modulated in blast cells after exposure to prednisolone and daunorubicin, which is partly consistent with data published by Gaipa
In addition to CD10, CD19, CD20, CD34 and CD58, we analysed other antigens whose modulation significance in patients with B-ALL has, to our knowledge, not yet been described. The experiment allowed us to assess shifts in the expression of CD27, CD45, CD66c and CD137.
Lower expression of CD45, an essential modulator of signal transduction pathways in blast cells, is associated with a higher rate of complete remissions at day 29 of B-ALL treatment and high expression of CD45 was associated with unfavourable prognostic factors and worse event free survival of the disease.10,16,26,38 In our experiment, CD45 was down-modulated after exposure to increasing concentrations of daunorubicin, and a dose-dependent up-modulation was observed after the addition of vincristine to the blast cells. Since it was shown that CD45 expression is higher in immature pro-B-ALL than in pre-B-ALL we could speculate that vincristine causes modulation of antigen expression towards the immunophenotype of pro-B-ALL.16 Additionally, it has been show that increased CD45 expression in blast cells is associated with decreased cell proliferation and treatment resistance but to determine the importance of changes in CD45 expression after exposure to daunorubicin and vincristine, further test would be needed.16
CD137 is known to be present in T lymphocytes and natural killer cells.39 The antigen is also expressed in human B lymphocytes, particularly on activated B cells.28 Despite the low expression of CD137 (data not shown), we were able to detect an up-modulation of the antigen, but only after exposure to daunorubicin. Zhang
CD27, a member of the tumour necrosis factor receptor superfamily, is mainly present on T cells, natural killer cells and thymocytes.40 It can also be detected on the surface of memory B cells as well as on high risk B-ALL with Philadelphia chromosome, cytokine receptor-like factor 2 (CRLF2) rearrangement or in B-other ALL with unknown or not classifying genetic aberrations, where it is additional poor prognostic factor.11,13,41 In oncology, CD27 is important in induction of cytotoxic T lymphocyte activation and CD27 agonists were proven to induce antitumor immunity.13,42 Additionally Vitale
Patients diagnosed with Philadelphia chromosome positive ALL are usually treated with a combination of tyrosine kinase inhibitors and standard chemotherapy.44 CD66c is the myeloid antigen commonly expressed on malignant B lymphoblasts, has no clear prognostic significance but is associated with important genetic alterations such as the presence of a Philadelphia chromosome, hyperdiploidy, hypodiploidy and CRLF2-positivity in B-ALL.17,18 After analysing the effects of chemotherapy, we found that CD66c expression was mostly low but quantifiable and that none of the drugs had an impact on its expression. Importantly, none of the included patients had Philadelphia chromosome positive ALL. Since the expression of CD66c was stable after the exposure to all tested drugs, CD66c could be a good candidate to MRD detection in patients with CD66c positive B-ALL.
The present
Moreover, according to our results, cytotoxic drugs trigger other changes in the expression of antigens that could be prognostically important. However, further analyses with a larger number of patients are required to determine the clinical significance of these changes.