Immunosuppressive monocytes (CD14+/HLA-DRlow/-)

Medical Oncology (2018) 35:36 https://doi.org/10.1007/s12032-018-1092-9

SHORT COMMUNICATION

Immunosuppressive monocytes (CD14+/HLA‑DRlow/−) increase in childhood precursor B‑cell acute lymphoblastic leukemia after induction chemotherapy D. S. Lima1,2 · R. P. G. Lemes2 · D. M. Matos3 Received: 18 January 2018 / Accepted: 31 January 2018 © Springer Science+Business Media, LLC, part of Springer Nature 2018

Abstract In tumor microenvironment, immunosuppression is a common event and results from the inhibition of activated immune cells and generation of cells with immunosuppressive capacity, as some subtypes of monocytes. The aim of this study was to evaluate the presence of immunosuppressive CD14+/HLA-DRlow/− monocytes in pediatric patients with the diagnosis of B-cell acute lymphoblastic leukemia (B-ALL) and, moreover, verify whether the chemotherapeutic treatment has any effect on these cells. Peripheral blood (PB) and bone marrow (BM) samples were collected from 15 untreated pediatric patients. The presence of C D14+/HLA-DRlow/− monocytes was evaluated at diagnosis and in the end of induction chemotherapy by flow cytometry. CD14+/HLA-DRlow/− monocytes increase was observed in 60% (9/15) of the patients at the end of the induction therapy. We were able to detect an increase in CD14+/HLA-DRlow/− monocytes values in BM and PB samples of pediatric patients with B-ALL. This increase was observed in the end of induction chemotherapy, which leads us to believe that these changes probably could have been induced by the inflammatory process engendered by the cytotoxic treatment or by drugs used in the chemotherapy treatment. This finding may be useful to guide new therapeutic approaches contemplating immunomodulatory drugs that act in the depletion of immunosuppressive monocytes. Keywords Acute lymphoblastic leukemia · Immunosuppressive monocytes · Flow cytometry

Introduction Acute lymphoblastic leukemia (ALL) is an abnormal proliferation and accumulation of clonal progenitor cells compromised with the differentiation of lymphoid cells in the bone marrow [1]. The immunosuppressive state observed in tumor microenvironment plays an important role in cancer initiation, progression and therapeutic failure [2]. CD14+/HLADRlow/− monocytes mediate immunosuppression through a range of mechanisms as, for example, release of interleukin-10 [3] and induction of T-cell regulatory populations [4]. * D. M. Matos [email protected] 1

Oncohematology Section, Albert Sabin Children Hospital, Ceará, Brazil

2

Department of Clinical and Toxicological Analysis, Federal University of Ceará, UFC, Ceará, Brazil

3

Flow Cytometry Section, Clementino Fraga Laboratory, Ceará, Brazil

The presence of immunosuppressive C D14 +/HLADRlow/− monocytes was previously described in malignant solid tumors as melanoma and prostate cancer [3, 5], and also in hematologic cancers as multiple myeloma [5], nonHodgkin lymphoma [6] and chronic lymphocytic leukemia [7]. Here, we evaluated the presence of C D14 + /HLADRlow/− monocytes in pediatric patients with the diagnosis of precursor B-ALL and, besides, we sought for any effect of chemotherapeutic treatment with regard to this population of monocytes.

Patients and methods Patients From August 2015 to May 2017, peripheral blood (PB) and bone marrow (BM) samples were collected from 33 untreated pediatric patients who fulfill the World Health Organization diagnostic criteria for de novo precursor

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B-ALL. Out of 33 initial patients recruited, 18 died or there was insufficient sample from bone marrow aspirate at the end of induction chemotherapy protocol. Thus, 15 patients were further stratified as high or low risk of relapse, based on the following criteria: age, initial white blood cells count, CNS involvement, cytogenetic and molecular profile [8]. The induction chemotherapy protocol was performed according to the recommendations of the Brazilian Cooperative Group for Childhood ALL Treatment (GBTLI-2009) [8]. Briefly, the induction chemotherapy for low risk B-ALL patients includes: dexamethasone (6 mg/m2), prednisone (60 mg/m2), vincristine (1.5 mg/m2), daunorubicin (25 mg/ m2), l-asparaginase (5000 U/m2) and, in presence of clinical indication, MADIT (intrathecal methotrexate, cytarabine and dexamethasone) (10–12 mg/m2, 20–24 mg/m2, 2 mg/m2, respectively). Alternatively, for high-risk B-ALL patients, the induction chemotherapy includes: prednisone (60 mg/ m2), vincristine (1.5 mg/m2), daunorubicin (40 mg/m2 per day), l-asparaginase (10,000 U/m2), cyclophosphamide (500 mg/m2) and, in presence of clinical indication, MADIT (intrathecal methotrexate, cytarabine and dexamethasone) (10–15 mg/m2, 20–30 mg/m2, 2 mg/m2, respectively). Informed consent was obtained from the parents of all the patients included, and, moreover, the study was approved by the Albert Sabin Hospital local ethical committee.

Cytogenetics and molecular analysis The G-band cytogenetic analysis was performed according to the recommendations elsewhere [9]. The molecular analysis of fusion genes expression was performed by reverse transcriptase-polymerase chain reaction (RT-PCR) technique using SuperScript™ III One-Step RT-PCR System with Platinum™ Taq High Fidelity DNA Polymerase kit (Invitrogen™), following the recommendations described by the manufacturer. All patients were evaluated for the presence of AF4/MLL, BCR/ABL1 p190 and p210, E2A/PBX1 and ETV6/RUNX1 fusion genes.

CD14+/HLA‑DRlow/− monocytes detection The presence of C D14+/HLA-DRlow/− monocytes was evaluated—at diagnosis (D0, day zero) and in the end of induction chemotherapy protocol (D35, day thirty five of treatment)— in PB and BM samples, by flow cytometry, using the following anti-human monoclonal antibodies conjugated with fluorochromes: CD14 (PE), CD19 (FITC), CD45 (PerCP) and HLA-DR (FITC). All monoclonal antibodies were purchased from BD Biosciences® (San Jose, CA, USA). The acquisition was performed using FACSCalibur™ flow cytometer, and the analysis was done using the CellQuest Becton–Dickinson® software. Initially, a region (R1) was defined in CD45 versus internal complexity (SSC)

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dot-plot to determine the cells of lymphomonocytic lineage. A second region (R2) was defined in CD14 versus internal complexity (SSC) dot-plot to specifically delineate the monocytic lineage. The next step was to evaluate the dot-plot HLA-DR versus CD14 with the purpose of quantifying the CD14+/HLA-DRlow/− monocytes (Fig. 1). For all analysis, 100,000 events were acquired. Based on minimal residual disease studies performed in ALL patients and the accurate quantitation of rare events strategy, we used a minimum of 40 clustered events characterized by both CD14 positivity and HLA-DR with low/negative expression in order to consider a sample containing an immunosuppressive monocyte population [10].

Results Sixty percent (9/15) of patients were stratified as high risk and 40% (6/15) as low risk. Forty-six percent (7/15) of patients showed aberrant karyotype that included numerical and/or structural alterations, and 53% (8/15) of patients presented positivity for one of the three fusion genes E2A/ PBX1, ETV6/RUNX1 or AF4/MLL. None of the patients was positive for BCR/ABL1 fusion gene. Table 1 shows patients’ clinical and biological data, as well as the percentages and absolute values of C D14+/HLAlow/− DR monocytes in peripheral blood and bone marrow, at diagnosis (D0) and after the initial chemotherapy protocol (D35). There was an expansion of C D14+/HLA-DRlow/− monocytes observed in 60% (9/15) patients when the values of CD14+/HLA-DRlow/− cells at the final of induction chemotherapy (D35) were compared to those of the diagnosis (D0). Out of this total, approximately 56% (5/9) presented the expansion in BM and PB concomitantly, while 44% (4/9) presented the expansion in BM or PB only. Three patients demonstrated CD14+/HLA-DRlow/− monocytes at diagnosis that were confined to PB samples.

Discussion CD14+/HLA-DRlow/− monocytes have important modulatory action on the interaction between immune system and malignant cells [11]. Here, we were able to detect an increase in CD14+/HLADRlow/− monocytes between D0 and D35 of chemotherapy treatment, in BM and/or PB samples of 60% (9/15) pediatric patients with the diagnosis of B-ALL. The fact that the increase was observed in the end of induction chemotherapy leads us to believe that this change could probably have been induced by the inflammatory process produced by cytotoxic chemotherapy, by the effect of some specific drugs


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Fig. 1 Flow cytometric analysis in BM of patient number 1 at D0 and D35, respectively. a The first dot-plot (CD45 vs. SSC) delimited the lymphomonocytic region (gate R1). The second dot-plot (CD14 vs. SSC) delimited the monocytic region (gate R2). The third dot-plot (HLA-DR vs. CD14) delimited the population of interest, namely

CD14+/HLA-DRlow/− monocytes (circle). b The same strategy of analysis was performed, but now in the end of induction therapy (D35). Notice the presence of 5.9% of CD14+/HLA-DRlow/− monocytes at the final of induction chemotherapy (D35)

included in the chemotherapy protocol, or by both. Thus, in a previous study, Mougiakakos et al. [12] showed that the frequency of circulating CD14+/HLA-DRlow/− monocytes was significantly increased after allogeneic hematopoietic stem cell transplantation, especially in patients with more severe acute graft-versus-host disease. In animals models, the treatment with cyclophosphamide has induced the expansion of monocytic immunosuppressive myeloid cells (CD11b+Ly6ChiCCR2hi), which are not equal, but still equivalent to human immunosuppressive CD14+/ HLA-DRlow/− monocytes, in mice with advanced B-cell lymphoma or lung metastasis of colon cancer [13]. Whether cyclophosphamide or possibly other cytotoxic drugs used in treatment protocols of pediatric patients with B-ALL could be responsible for the expansion of CD14+/HLADRlow/− monocytes observed in our patients and, moreover, whether this expansion would contribute to tumor progression and/or therapeutic failure is uncertain, and, thus, more studies with longer time of follow-up are required. In our study, three patients (number 8, 12 and 13) (Table 1) demonstrated C D14+/HLA-DRlow/− monocytes at diagnosis that were confined to PB samples. We have no

definitive explanation for this phenomenon. However, we can speculate that the non-detection of the C D14+/HLAlow/− DR monocytes in BM samples of these three patients can probably be justified by the fact that, notwithstanding the CD14+/HLA-DRlow/− monocytes accumulate at the tumor site (medullar microenvironment), these cells are rapidly recruited by tumor cells and enter in a process of differentiation which transform them in a specific type of macrophages, namely tumor-associated macrophages (TAMs), as suggested by recent data [14]. Intriguingly, we verified uncommon C D14 +/HLADRlow/− monocytes kinetics in two patients. Namely, the values of CD14+/HLA-DRlow/− monocytes observed at diagnosis in patients 8 and 13 declined after initial induction therapy. Curiously, a similar finding was detected in patients with the diagnosis of chronic lymphocytic leukemia, where a reduced frequency of C D14+/HLA-DRlow/− monocytes was seen after the chemotherapeutic treatment [7]. CD14+/HLA-DRlow/− cells are a subtype of human myeloid-derived suppressor cells (MDSCs) which are mainly described as the monocytic subpopulation [15]. Recently, Liu et al. [16], analyzing another subtype of human MDSC

13

13

M

10

3y

5y

1y

2y

3y

3y

1y

6y

7y

17y

1y

3y

15y

9y

9y

Age (years)

Death

a

AM Absence of metaphases

RT-PCR

3

0%/(0 cells/mm ) 0%/(0 cells/mm3)

2.1%/(73 cells/mm3)

3


0%/(0 cells/mm3)

Negative AF4/MLL positive

ETV6/RUNX1 positive

E2A/PBX1 positive

Negative

Negative

46,XX[14]

ETV6/RUNX1 positive Negative

Negative

46,XY[18]

ETV6/RUNX1 positive

59,XX,+1,+4,+5,+6,+9,+11, Negative +12,+14,+16,+17,+18,+21, +22[12]/46,XX[3]

46,XX[18]

46,XX[16]

60.6%/(16.241 cells/ mm3) 7.5%/(1.260 cells/mm3) 6.9%/(227 cells/mm3) 0.9%/(48 cells/mm3)

0%/(0 cells/mm3) 0%/(0 cells/mm3) 9.9%/(118 cells/mm3) 6.9%/(110 cells/mm3)

0%/(0 cells/mm ) 0.6%/(78 cells/mm3)


3

4.7%/(583 cells/mm )

0%/(0 cells/mm )

3

3

0%/(0 cells/mm3)

7.9%/(126 cells/mm3) 3

23.6%/(5.475 cells/ mm3)

0%/(0 cells/mm )

3

0%/(0 cells/mm3)

0%/(0 cells/mm3)

AF4/MLL positive

54,XY,+X,+6,+14,+17,+18,+ Negative 21,+21,+mar[10]/46,XY[10]

AM

46,XY,inv(9)(p12q13)c[20]

46,XX[20]

AM

80,XXY,+X,+1,+2,+3,t(4;11) (q21;q23),+8,+12,+17,−18, +19,+21,+22,+mar1,+mar2 [9]/46,XY[3]

55,XY,+X,+4,+6,+8,+9,+10, +13,+21+22[9]/46,XY[3]

46,XX,t(4;11)(q21;q23)[12]

3

13%/(2.860 cells/mm3) 2.3%/(62 cells/mm )

3

0%/(0 cells/mm3)

0%/(0 cells/mm3)

0%/(0 cells/mm )

3

0%/(0 cells/mm3)

0%/(0 cells/mm3)

0%/(0 cells/mm3)

0%/(0 cells/mm3)

0%/(0 cells/mm )

3

0%/(0 cells/mm3)

0%/(0 cells/mm )

3

0%/(0 cells/mm3)

0%/(0 cells/mm3)

0%/(0 cells/mm )

3

0%/(0 cells/mm3)

0%/(0 cells/mm )

3

0%/(0 cells/mm3)

22

10

10

10

12

14

15

2a

20

20

21

0%/(0 cells/mm3)

0%/(0 cells/mm3)

0%/(0 cells/mm3)

3

6

6

9.9%/(1.603 cells/mm3) 7

5.7%/(296 cells/mm3)

25.4%/(6.959 cells/ mm3)

2.2%/(143 cells/mm3)

0%/(0 cells/mm3)

16%/(848 cells/mm3)

0%/(0 cells/mm3)

0%/(0 cells/mm3)

0%/(0 cells/mm3)

0%/(0 cells/mm3)

0%/(0 cells/mm3)

5.9%/(944 cells/mm3)

Peripheral blood CD14+/HLA-DRlow/− monocytes Bone marrow CD14+/HLA-DRlow/− monocytes Follow-up (months) D0 (%)/(mm3) D35 (%)/(mm3) D0 (%)/(mm3) D35 (%)/(mm3)

0%/(0 cells/mm )

46,XY,der(19)t(1;19)(q23;p13) E2A/PBX1 positive [18]

47,XY,t(1;19)(q23;q13),+8[16] E2A/PBX1 positive

Cytogenetics analysis

D35 = Day 35 of treatment (final of induction therapy)

Low risk

Low risk

Low risk

Low risk

Low risk

High risk

High risk

High risk

High risk

High risk

High risk

Low risk

High risk

High risk

High risk

Risk stratification

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D0 = Day zero of treatment

M

F

9

15

M

8

F

F

7

14

M

6

F

M

5

13

M

4

F

F

3

F

M

2

11

M

1

12

Sex

Patient Id

Table 1 Patients data and C D14+/HLA-DRlow/− monocytes population in PB and BM

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(granulocytic-MDSC), showed that these cells were significantly elevated in both the peripheral blood and bone marrow of patients with B-ALL. Taken together, Liu et al. and our findings suggest that immunosuppressive cells, being of granulocytic or monocytic lineage, are present in patients with B-ALL and, hence, we can speculate that the increased numbers of MDSCs might contribute to the immunosuppressive microenvironment inside the bone marrow. In summary, we demonstrated the presence and expansion of CD14+/HLA-DRlow/− monocytes, in the bone marrow and peripheral blood, of patients with the diagnosis of B-cell acute lymphoblastic leukemia. As far as we know, this is the first study investigating the presence of C D14+/HLAlow/− DR monocytes in patients with the diagnosis of B-cell acute lymphoblastic leukemia and, moreover, the influence of chemotherapy treatment on these cells, provided that we studied the C D14+/HLA-DRlow/− population in two different moments of treatment, namely at the diagnosis and at the end of induction chemotherapy regimen. This finding may be useful to guide new therapeutic approaches in the future, since that, in recent years, MDSCs are considered as a potential target in hematological malignancies to enhance the effects of currently used immune modulating agents [15]. Acknowledgements The authors thank the Clementino Fraga Laboratory for funding the research. The authors thank Débora Yasmin de Sousa and Thayna Nogueira dos Santos for technical assistance with flow cytometry.

5. 6. 7.

8.

9.

10.

11.

12.

13.

Compliance with ethical standards Conflict of interest The authors declare no conflict of interest.

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