The Egyptian Journal of Haematology

ORIGINAL ARTICLE
Year
: 2014  |  Volume : 39  |  Issue : 4  |  Page : 183--189

Cerebrospinal fluid and serum levels of monocyte chemoattractant protein 1 in acute leukemia patients: correlation to other prognostic factors


Mohammad I Ahmed1, Dalia A Nafe2, Maha Y Kamal3,  
1 Department of Clinical Pathology, Alexandria, Egypt
2 Hematology Unit, Department of Internal Medicine, Alexandria, Egypt
3 Hematology Unit, Department of Pediatric, Alexandria, Egypt

Correspondence Address:
Mohammad I Ahmed
Department of Clinical Pathology, Alexandria 21527
Egypt

Abstract

Background Acute leukemia (AL) is a malignancy with accumulation of blasts in the bone marrow (BM). The blast cells enter into the peripheral blood stream and secondary localized extramedullary sites. The regulation of this process has not been clearly explained so far. Objective The aim of this study was to evaluate the cerebrospinal fluid (CSF) and serum levels of monocyte chemoattractant protein 1 (MCP1) in AL patients with correlation to other prognostic factors and tumor load. In a case-control study, 80 de-novo AL patients and 30 healthy age-matched controls were included. All patients were subjected to thorough history taking, full physical examination, and laboratory investigations such as complete blood count, BM examination, and flow cytometry (by enzyme-linked immunosorbent assay method). Results MCP1 was significantly elevated in both blood and CSF in AL patients in comparison with the control group (P < 0.05). In AL patients, there was a significant positive correlation between MCP1 level in both blood and CSF and the following parameters: hepatomegaly, splenomegaly, lymphadenopathy, purpura, fever, platelet count, white blood cells, and blast % in peripheral blood and BM (P < 0.05) were observed. There was no correlation between MCP1 in blood and CSF and Hb, lactate dehydrogenase and first hour of erythrocyte sedimentation rate. Conclusion Significant increase in the CSF and blood levels of MCP1 level was observed in AL patients compared with the control group. This may be related to extramedullary leukemic infiltration, tumor load, and disease activity in these patients. Egyptian J Haematol 39:-0 © 2014 The Egyptian Society of Haematology.



How to cite this article:
Ahmed MI, Nafe DA, Kamal MY. Cerebrospinal fluid and serum levels of monocyte chemoattractant protein 1 in acute leukemia patients: correlation to other prognostic factors.Egypt J Haematol 2014;39:183-189


How to cite this URL:
Ahmed MI, Nafe DA, Kamal MY. Cerebrospinal fluid and serum levels of monocyte chemoattractant protein 1 in acute leukemia patients: correlation to other prognostic factors. Egypt J Haematol [serial online] 2014 [cited 2019 Dec 9 ];39:183-189
Available from: http://www.ehj.eg.net/text.asp?2014/39/4/183/153942


Full Text

 Introduction



Acute leukemia (AL) is the most common form of hematological malignancies, with ALL being more common than acute myeloid leukemia (AML). ALL is more common among children group with cure rates exceeding 80%. However, up to one quarter of children relapse; the cure rates for childhood AML are substantially lower. AL is less common in adults, and more commonly myeloid than lymphoid, and generally carries a worse prognosis than in children [1].

Patients with AL present with symptoms due to organomegaly cytopenias, lymphadenopathy, and bone pains. Skeletal manifestation presenting in the form of bone pains and refusal to walk may be seen in 40-60% of children [2].

AL is a diverse group of malignancies with a range of clinical presentation, prognosis, and preferred treatment protocols. Historical classification systems relied predominantly on morphologic and cytochemical features; however, currently, immunophenotypic, cytogenetic, and molecular data are incorporated to define clinically relevant diagnostic categories [3].

The blast cells enter into the peripheral blood (PB) stream and secondary localized extramedullary sites. The regulation of this process has not been clearly explained so far, but interactions between some chemokines and their specific receptors could be one of the mechanisms responsible for such kind of migration [4].

Cytokines and adhesion molecules have been studied in many pathological states including hematological malignancies [5],[6],[7] and AL, both myeloid (AML) and lymphoblastic (ALL) [8],[9]. Alterations in this interacting functional network may have direct effect on the malignant cells or have indirect effect on leukemogenesis through altered functions of bone marrow (BM) stromal elements [10],[11].

 Materials and methods



This study was conducted on two groups. Group 1 consisted of de-novo AL 80 patients. They were further subclassified as ALL (group 1a) and AML (group 1b). Group 2 consisted of 30 healthy people with matched age and sex.

All patients were admitted to the Hematology Unit of Internal Medicine and Pediatric Departments, Alexandria University Hospital. These cases were diagnosed on clinical presentation, morphology and cytochemical stains, immunophenotyping, and molecular techniques if needed. Informed consent was taken from all patients and approval of the ethical committee was taken.

All patients were subjected to the following:

(1) Detailed history: with special emphasis on age, sex, presence of leukemia, associated symptoms (fever, easy fatigability, bleeding tendency, and bone aches), symptoms of central nervous system (CNS) involvement (vomiting, headache, blurring of vision), and duration of the disease.

(2) Clinical examination: pallor, petechiae and purpuric eruptions, hepatomegaly, splenomegaly, lymphadenopathy, gum hypertrophy, signs of infection, skin infiltration, and CNS involvement.

(3) Abdominal ultrasonography: for the presence of hepatosplenomegaly.

(4) Laboratory investigation:

(a) Routine laboratory investigation: complete blood count, liver profile, renal profile, erythrocyte sedimentation rate (ESR) and lactate dehydrogenase (LDH). Cerebrospinal fluid (CSF) examination for blast cell infiltration [12].

(b) BM aspiration and examination of Leishman-stained smears and cytochemical stain: to confirm the diagnosis by Sudan Black-B, myeloperoxidase, and periodic acid Schiff (Sigma-Aldrich, St. Louis, USA) [12].

Immunophenotyping of all cases for diagnosis by flow cytometry using the panel for AL [12]:

(1) Pan leukocytic marker: CD45.

(2) Hematopoietic progenitor cells markers: CD34 and HLA-DR.

(3) Myeloid cell markers: CD13, CD33 and CD14, and MPO.

(4) Lymphoid cell markers: B-cell markers (CD10, CD19, and CD22), T-cell markers (CD3, CD4, CD5, CD2, and CD7).

Monocyte chemoattractant protein 1 (MCP1) by enzyme-linked immunosorbent assay [13].

Statistical analysis

The data were collected and entered into the personal computer. Statistical analysis was performed using Statistical Package for Social Sciences (SPSS version 20, Chicago, IL, USA) software. For the arithmetic mean and SD for categorized parameters, c2 -test was used, whereas for numerical data for more than two groups analysis of variance test was used. To find the association between two variables, spearman correlation coefficient test was used. The level of significant was 0.05.

 Results



The study included 80 patients of newly diagnosed de-novo AL (group 1). They were 60 ALL patients (group 1a) and 20 AML patients (group 1b). Thirty healthy individuals were included in the study as control group (group 2).

With regard to age and sex

The age of ALL patients (group 1a) ranged from 1.5 to 59.0 years, with a mean value of 25.1 ± 14.6. AML patient (group 1b) age ranged from 3.0 to 49.0 years with a mean value of 34.1 ± 15.22 [Table 1]. The age of the patients in the control group ranged from 3.0 to 54.0 years, with a mean value of 28.5 ± 16.2. There was no statistical difference between both the groups in terms of age (P = 0.108).{Table 1}

Of the 60 ALL patients (group 1a), 41 (68%) were male, and 19 (32%) were female. Of the 20 AML patients (group 1b), 12 (60%) were male, and eight (40%) were female. Of all the subjects in the control group, 20 (67%) were male, and 10 (33%) were female. There was no statistical difference between both the groups in terms of sex (P = 0.235).

Clinical findings

Hepatomegaly was observed in 41/60 (68.3%) ALL patients and in 12/20 (60.0%) AML patients [Table 2]. Splenomegaly was observed in 48/60 (80.0%) ALL patients and in 11/20 (55.0%) AML patients. Purpura was observed in 50/60 (83.3%) ALL patients and in 7/20 (35.0%) AML patients. Lymphadenopathy was observed in 54/60 (90.0%) ALL patients and in 3/20 (15.0%) AML patients. Fever was observed in all 60 (100.0%) ALL patients and in 18/20 (90.0%) AML patients. Gum hypertrophy was observed in none of the ALL patients (0.00%) and in 2/20 (20.0%) AML patients. Skin involvement was not observed in either the ALL patients (0.00%) and or in any of the AML patients (0.00%). CSF infiltration was observed in 4/60 (6.7%) ALL patients and in none of the AML patients (0.00%). Testicular infiltration was observed in 3/60 (5.0%) ALL patients and in none of the AML patients (0.00%).{Table 2}

Laboratory investigations

Hemoglobin level

0In the patient group, Hb ranged from 3.3 to 12.3 g/dl, with a mean value of 6.25 ± 2.86 [Table 3]. In the control group, Hb ranged from 11.5 to 14.8 g/dl, with a mean value of 12.65 ± 1.06. There was a significant decrease in Hb level in the ALL patient group compared with the control group (t = 5.65, P = 0.001).{Table 3}

Platelet count

Platelet count in the ALL patient group ranged from 18.0 to 120.0/mm 3 , with a mean value of 45.6 ± 20.13. In the control group, platelet count ranged from 190 to 238.0/mm 3 , with a mean value of 221.3 ± 12.65. There was a statistical difference between both the groups in terms of platelet count (t = 7.35, P = 0.001).

White blood cells

In the patient group, white blood cell (WBC) count ranged from 12.5 to 527.22/mm 3 , with a mean value of 214.6 ± 85.2. In the control group, WBC count ranged from 4.0 to 10.1/mm 3 , with a mean value of 6.25 ± 1.85. There was a significant increase in WBC count in the ALL patient group compared with the control group (t = 18.65, P = 0.0001).

Erythrocyte sedimentation rate

In the patient group, first hour ESR ranged from 38 to 130 mm/h, with a mean value of 85.6 ± 22.9. In the control group, first hour ESR ranged from 5 to 18 mm/h, with a mean value of 10.36 ± 2.85. There was a significant increase in the ESR in the ALL patient group compared with the control group (t = 17.65, P = 0.0001).

Lactate dehydrogenase level

In the patient group, LDH level ranged from 640 to 1687 U/l, with a mean value of 1085 ± 205.3. In the control group, LDH level ranged from 216 to 380 U/l, with a mean value of 290.5 ± 45.8. There was a significant increase in LDH level in the ALL patient group compared with the control group (t = 22.6, P = 0.0001).

FAB classification and immunophenotyping of patients

(1) FAB of ALL patients: L1 was found in 46 (76.7%) cases, L2 in 10 (16.6%) cases, and L3 in four (6.7%) cases of ALL. Immunophenotyping showed that there were 51 (85.0%) cases of precursor B-ALL (early pre-B, common ALL, patients pro-B), four (6.7%) cases of mature B-ALL, and five (8.33%) cases of T-ALL.

(2) FAB and immunophenotyping of AML patients showed: M0 (0.00%), M1 (20.0%), M2 (30.0%), M3 (5.00%), M4 (40.0%), M5 (5.00%), M6 (0.00%), and M7 (0.00%).

Blast percentage in peripheral blood and bone marrow

In ALL (group 1a) patients, blast % in PB ranged between 8 and 83%, with a mean of 52.6 ± 22.6 [Table 4]. Blast % in BM ranged between 30 and 91%, with a mean of 45.2 ± 12.6.{Table 4}

In AML (group 1b) patients, blast % in PB ranged between 2 and 68%, with a mean of 38.9 ± 16.5. Blast % in BM ranged between 68 and 91%, with a mean of 75.6 ± 10.3.

Bone marrow cellularity

In the ALL group (group 1a), hypercellular BM was observed in 57 (95.0%) patients, and normocellular BM was observed in three (5.0%) patients [Table 5]. In the AML group (group 1b), hypercellular BM was observed in 18 (90.0%) patients, and normocellular BM was observed in two (10.0%) patients. There was no significant difference between the two patient groups (P = 0.423).{Table 5}

Blood monocyte chemoattractant protein 1 level in patient and control groups

In the ALL patients (group 1a), MCP1 level ranged from 290 to 1027 U/l, with a mean value of 452.6 ± 115.3 [Table 6]. In the AML patients (group 1b), MCP1 level ranged from 260 to 1310 U/l, with a mean value of 625.3 ± 154.6. In the control group, MCP1 level ranged from 205 to 630 U/l, with a mean value of 325.6 ± 85.9. There was a significant increase in blood MCP1 level in the ALL (group 1a) AML (group 1b) groups compared with the control group (P = 0.0052).{Table 6}

Cerebrospinal fluid monocyte chemoattractant protein 1 level in patient and control groups

In ALL patients (group 1a), MCP1 level ranged from 80 to 420 pg/ml, with a mean value of 240.2 ± 95.2 [Table 6]. In AML patients (group 1b), MCP1 level ranged from 101 to 510 pg/ml, with a mean value of 321.2 ± 89.5. In the control group, MCP1 level ranged from 78 to 110 pg/ml, with a mean value of 85.9 ± 8.68. There was a significant increase in CSF MCP1 level in the ALL (group 1a) AML (group 1b) patient groups compared with the control group (P = 0.001).

Correlation of monocyte chemoattractant protein 1 in blood and cerebrospinal fluid (groups 1a and 1b) with different parameters

In ALL and AML patients, there was a significant positive correlation between MCP1 level in both blood and CSF and the following parameters: hepatomegaly, splenomegaly, lymphadenopathy, purpura, fever, platelet count, WBCs, blast % in PB and BM, gum hypertrophy, CSF infiltration, and testicular infiltration (P<0.05) [Table 6]. There was no correlation between MCP1 in blood and CSF and Hb, LDH, and first hour ESR.

 Discussion



Chemokines play an important role in leukocyte mobilization, hematopoiesis, and angiogenesis. Tissue-specific expression of particular chemokines also influences tumor growth and metastasis [14].

Cytokines and adhesion molecules have been studied in many pathological states including hematological malignancies [15] and AL, both myeloid (AML) and lymphoblastic (ALL) [16]. Alternation in this interacting functional network may have direct effect on the malignant cells or have indirect effect on leukemogensis through altered functions of BM stromal elements [17],[18].

The MCP1 is a member of the C-C chemokine family, and a potent chemotactic factor for monocytes. MCP1 is of particular relevance to cancer invasion and metastasis as it is overexpressed in a variety of cancer types, including glioma, ovarian, lung, breast, and prostate cancers [19]. MCP1-mediated macrophage infiltration is known to promote tumor progression in various cancers, as MCP1 is known to play a crucial role in tumor tissue inflammatory response by activating and inducing the infiltration of macrophages and regulating adhesion factors [16].

The aim of this study was to evaluate the CSF and serum levels of MCP1 in AL patients with correlation to other prognostic factors and tumor burden.

In our study, there was a significant increase in blood MCP1 (CCL2) level in AL patients compared with the control group (P = 0.001).

Going with our results, Mazur et al. [20] evaluated the plasma level of CCL2 in 65 adult patients taken before chemotherapy and in complete remission measured by enzyme-linked immunoassay. The control group consisted of 15 healthy subjects. They found that in AML patients mean baseline CCL2 level was significantly higher than in normal controls: 365, 26 ± 5, 62 pg/ml versus 265, 56 ± 5, 48 pg/ml, respectively (P < 0.01). They demonstrate increased mean CCL2 plasma level in untreated patients with AML.

Horacek et al. [21] analyzed serum samples at the diagnosis of ALL (active leukemia) and healthy subjects (blood donors), circulating levels of 17 cytokines and five adhesion molecules. MCP1 level was significantly increased in ALL patients compared with the control group (433.99 ± 328.59 vs. 153.25 ± 53.60 ng/l; P<0.01). They concluded that their results indicate that serum levels of specific cytokines and adhesion molecules are significantly altered in patients with newly diagnosed ALL, reflecting activity of the disease.

Mazur et al. [20] evaluated the plasma level of CCL2 in patients with AML. Plasma samples from 65 adult patients with AML taken before chemotherapy and in complete remission were measured by enzyme-linked immunosorbent assay. They demonstrated increased mean CCL2 plasma level in untreated patients with AML. Significantly lower plasma level of CCL2 was observed in patients with M4 and M5 AML subtypes according to FAB classification. In the AML group, chemotherapy did not reduce CCL2 plasma level.

In our AL patients, there was a significant increase in CSF MCP1 level compared with the control group (P = 0.001). Eisenkraft et al. [22] studied 19 children with ALL and without known CNS involvement. CSF samples were aliquoted at different stages of therapy (diagnosis, induction, and maintenance). They concluded that CNS involvement in ALL was associated with significantly higher levels of MCP1 during therapy. This significant rise in MCP1 levels might be one of the mechanisms involved in the regulation of CNS leukemia.

In our study, in AL patients, there was significant positive correlation between MCP1 level in both blood and CSF and the following parameters: hepatomegaly, splenomegaly, lymphadenopathy, gum hypertrophy, CSF infiltration, and testicular infiltration (P<0.05). That may be reflecting the role of MCP1 in progress of the leukemic cells and its infiltration extramedullary to different organs such as the lymph nodes, spleen, liver, gum, testicles, and CNS.

In addition, there was significant positive correlation between MCP1 level in both blood and CSF and purpura, fever, platelet count, WBC, blast % in PB and BM (P<0.05). This may be reflecting the role of MCP1 in disease activity and may be a bad prognostic factor. There was no correlation between MCP1 in blood, CSF, Hb, LDH, and first hour ESR.

In our study, there was a significant positive correlation between MCP1 level in both blood and CSF and tumor load (manifested by high total leukocytic count, BM cellularity, blast % in PB and BM, and extramedullary infiltrations).

In a study carried out by Matar et al. [23], in a subset of patients where chemokine analysis was performed (30 patients); only MCP1 levels at day 30 posthematopoietic cell transplantation were predictive of relapse out of the 42 biological markers tested. The seven of 30 patients who relapsed in this subset (23%) had higher mean level of MCP1 at day 30 (P = 0.007). MCP1 was predictive of leukemic relapse 82 days in advance on average before overt hematological relapse. Full chimerism (>95%) was detected at day 30 in 5/7 patients who relapsed in the biological marker group. They concluded that serum MCP1 levels in the early post-transplant period were predictive of relapse in the subset of patients where posthematopoietic cell transplantation biomarkers were available.

 Conclusion



There was a significant increase in the CSF and blood levels of MCP1 level in AL patients compared with the control group. This may be related to extramedullary leukemic infiltration, tumor load, and disease activity in these patients.

 Acknowledgements



Conflicts of interest

There are no conflicts of interest.

References

1Mullighan C. Genomic analysis of acute leukemia. Int Jnl Lab Hem 2009; 31 :384-397.
2 Sadawaite S, Jijina F, Nair CK, Seth S, Ghosh K An unusual presentation of pediatric acute lymphoblastic leukemia.Indian J Hematol Blood Transfus 2008; 24 :59-62.
3 Peters JM, Ansari MQ. Multiparameter flow cytometry in the diagnosis and management of acute leukemia. Arch Pathol Lab Med 2011; 135 : 44-54.
4 Mazur G, Wrobel T, Butrym A, Kapelko-Slowik K, Poreba R, Kuliczkowski K. Increased monocyte chemoattractant protein 1 (MCP-1/CCL-2) serum level in acute myeloid leukemia. Neoplasma 2007; 54 :285-289.
5 Bruserud Ø, Kittang AO. The chemokine system in experimental and clinical hematology. Curr Top Microbiol Immunol 2010; 341 : 3-12.
6 Mellgren K, Hedegaard CJ, Schmiegelow K, Müller K Plasma cytokine profiles at diagnosis in pediatric patients with non-Hodgkin lymphoma. J Pediatr Hematol Oncol 2012; 34 : 271-275.
7 Deeg HJ. Cytokines in graft-versus-host disease and the graft-versus-leukemia reaction. Int J Hematol 2001; 74 : 26-32.
8 Lowenberg B, Touw IP. Hematopoietic growth factors and their receptors in acute leukemia. Blood 1993; 81 : 281-292.
9 Kupsa T, Horacek JM, Jebavy L. The role of cytokines in acute myeloid leukemia: a systematic review. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 2012; 156 : 291-301.
10Konopleva MY, Jordan CT. Leukemia stem cells and microenvironment: biology and therapeutic targeting. J Clin Oncol 2011; 29 : 591-599.
11Reikvam H, Hatfield KJ, Fredly H, Nepstad I, Mosevoll KA, Bruserud Ø The angioregulatory cytokine network in human acute myeloid leukemia - from leukemogenesis via remission induction to stem cell transplantation. Eur Cytokine Netw 2012; 23 : 140-153.
12Lewis SM, Bain BJ, Bates I. Dacie and Lewis practical hematology. 10th ed. Churchill Livingstone, Philadelphia; 2006.
13Burtis CA, Ashwood ER. Fundamentals of clinical chemistry. 5th ed. St. Louis: Saunders/Elsevier ; 2000.
14Struyf S, Schutyser E, Gouwy M, Gijsbers K, Proost P, Benoit Y, et al. PARC/CCL18 is a plasma CC chemokine with increased levels in childhood acute lymphoblastic leukemia. Am J Pathol 2003; 163 :2065-2075.
15Gerard C, Rollins BJ. Chemokines and disease. Nat Immunol 2001; 2 : 108-115.
16Begg GS, Pepper DS, Chesterman CN, Morgan FJ. Complete covalent structure of human beta-thromboglobulin. Biochemistry 1978; 17 : 1739-1744.
17Schulz-Knappe P, Mägert HJ, Dewald B, Meyer M, Cetin Y, Kubbies M, et al. HCC-1, a novel chemokine from human plasma. J Exp Med 1996; 183 : 295-299.
18Detheux M, Ständker L, Vakili J, Münch J, Forssmann U, Adermann K, et al. Natural proteolytic processing of hemofiltrate CC chemokine 1 generates a potent CC chemokine receptor (CCR)1 and CCR5 agonist with anti-HIV properties. J Exp Med 2000; 192 : 1501-1508.
19Balkwill F. Cancer and the chemokine network. Nat Rev Cancer 2004; 4 : 540-550.
20Mazur G, Wróbel T, Butrym A, Kapelko-S³owik K, Poreba R, Kuliczkowski K. Increased monocyte chemoattractant protein 1 (MCP-1/CCL-2) serum level in acute myeloid leukemia. Neoplasma 2007; 54 :285-289.
21Horacek JM, Kupsa T, Vasatova M, Jebavy L, Zak P. Evaluation of serum levels of multiple cytokines and adhesion molecules in patients with newly diagnosed acute lymphoblastic leukemia using biochip array technology. Exp Oncol 2013; 35 :229-230.
22Eisenkraft A, Keidan I, Bielorai B, Keller N, Toren A, Paret G. MCP-1 in the cerebrospinal fluid of children with acute lymphoblastic leukemia. Leuk Res 2006; 30 :1259-1261.
23A Matar, MM Solh, AJ Copik, S Litherland, Alvin oliveras Almodovar, K Rathmann, Y Khaled. Relapse after allogenic hematopoietic stem cell transplantation (HCT) for acute myeloid leukemia (AML)/myelodysplastic syndrome (MDS) following intravenous busulfan/fludarabine based conditioning: outcomes and monocyte chemo-attractant protein-1 (MCP-1) as a predictive marker of relapse. ASH 2013 Annual Meeting Abstract 3325 (Poster Presentation), New Orleans, 2013.