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 Table of Contents  
ORIGINAL ARTICLE
Year : 2013  |  Volume : 38  |  Issue : 2  |  Page : 74-79

Prognostic value of chemokine CCL3 cellular mRNA expression and serum CCL3 level in lymphoma patients


1 Department of Clinical Pathology, Faculty of Medicine, Tanta University, Tanta, Egypt
2 Department of Pathology, Faculty of Medicine, Tanta University, Tanta, Egypt
3 Department of Oncology, Faculty of Medicine, Tanta University, Tanta, Egypt

Date of Submission14-Jan-2013
Date of Acceptance14-Feb-2013
Date of Web Publication20-Jun-2014

Correspondence Address:
Maaly M. Mabrouk
Department of Clinical Pathology, Faculty of Medicine, Tanta University, Tanta
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.7123/01.EJH.0000428062.52188.cc

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  Abstract 

Background

Chemokines are responsible for metastatic dissemination of cancers, including lymphomas. CCL3, a monocyte inflammatory protein-1 α, has been shown to be active as an inhibitor of primitive hematopoietic cell proliferation in vitro and in vivo. A dysfunction in this inhibitory process has been postulated to contribute to lymphomagenesis. The aim of this study was to measure the serum level of CCL3 and CCL3 mRNA in lymphoma patients compared with its expression during reactive lymphadenopathy; we also studied their effect on treatment response and survival of lymphoma patients.


Materials and methods

The study included 53 lymphoma patients and 20 reactive lymphadenopathy patients. The serum level of CCL3 was assessed using the enzyme-linked immunosorbent assay technique, and CCL3 gene expression was measured using real time PCR.


Results

There was a significant increase in the serum level of CCL3 in lymphoma patients (mean, 72.5±24.3 pg/ml) compared with reactive lymphadenopathy patients (mean, 31.5±12.6 pg/ml). Receiver–operator characteristic curve analysis showed a cutoff value of more than 55 pg/ml. There were positive and negative correlations with the lactate dehydrogenase level and time of follow-up (months), respectively. CCL3 expression was higher in lymphoma patients (90.5%) than in reactive lymphadenopathy patients (35%). Increased serum levels and expression of CCL3 were associated with treatment resistance. As regards survival, increased CCL3 expression was associated with decreased survival rate in lymphoma patients.


Conclusion

CCL3 serum levels or gene expression could be a valuable prognostic marker and may provide insight into creating a new therapeutic modality.

Keywords: CCL3, chemokine, lymphomas


How to cite this article:
Mabrouk MM, Ezzat A, Suliman GA, Aziz DA, Elfattah OA. Prognostic value of chemokine CCL3 cellular mRNA expression and serum CCL3 level in lymphoma patients. Egypt J Haematol 2013;38:74-9

How to cite this URL:
Mabrouk MM, Ezzat A, Suliman GA, Aziz DA, Elfattah OA. Prognostic value of chemokine CCL3 cellular mRNA expression and serum CCL3 level in lymphoma patients. Egypt J Haematol [serial online] 2013 [cited 2020 Jan 24];38:74-9. Available from: http://www.ehj.eg.net/text.asp?2013/38/2/74/134792


  Introduction Top


Chemokines are a large family of small soluble proteins that have been identified as attractants of blood leukocytes to sites of infection and inflammation and are stimulated by inflammatory cytokines, growth factors, and pathogenic stimuli 1,2.

Abnormalities in their production or signaling may have an important role in carcinogenesis 3,4 and hematolymphoid neoplasia 5.

Chemokines are classified into four groups according to the position of the first two cysteine residues in the amino acid sequence at the amino terminus. The C–C family of chemokines includes the monocyte chemoattractant protein (CCL2), macrophage inhibitory proteins (MIP)-1α (CCL3) and MIP-1β (CCL4), and RANTES (CCL5) 6.

CCL3 protein is released by most immune cells in response to various proinflammatory stimuli and is an important chemoattractant in cells essential for innate and adaptive immunity 6–7.

CCL3 expressed its biologic effects by binding to two related cell surface G protein-coupled receptors, chemokine receptor 1 (CCR1) and chemokine receptor 5 (CCR5, which initiate many cellular responses that regulate acute and chronic inflammation 8.

CCL3 is an active inhibitor of primitive hematopoietic cell proliferation in vitro and in vivo. A dysregulation of this inhibitory process has been postulated to contribute to leukemogenesis 9.

Several studies have reported the implication of chemokine signaling in cancer treatment response and metastasis through autocrine and paracrine mechanisms 10,11.

Lymphoma is the most common form of hematological malignancy; it represents 5.3% of all cancers and 55.6% of all blood cancers in the USA. Hodgkin’s lymphoma (HL), in particular, accounts for less than 1% of all cases of cancer in the USA 12.

Diffuse large B-cell lymphoma is the most common subtype of non-Hodgkin’s lymphoma. Less than half of the patients are cured with standard therapy for diffuse large B-cell lymphoma. This outcome reflects a heterogeneous group of tumors, with different genetic abnormalities and responses to therapy. The biological insights provided by molecular studies should allow for more targeted therapies to be developed, which will increase treatment choice and the possibility of tailored therapy in the future 13.

Tumor cells secrete chemokines and cytokines, attracting inflammatory cells and enhancing tumor growth and progression 14. Lymphadenopathy can also be present in both lymphoma and inflammation. However, lymph node metastases and clinical reactive lymph node enlargement differ in the profile of chemokines and their receptors expressed 15.

The aim of this study was to measure the serum level of CCL3, to determine CCL3 gene expression in lymphoma patients compared with reactive lymphadenopathy patients, and to determine their effect on treatment response and survival of lymphoma patients.


  Patients and methods Top


This study was carried out on 53 patients presenting with lymph node enlargement at the Clinical Oncology Department, Tanta University Hospital, Egypt. They were treated and followed up in this study during the period from October 2009 to October 2012. They had generalized lymph node enlargement. Formalin-fixed and paraffin-embedded sections were prepared for the following procedures:

  1. Routine hematoxylin and eosin (H&E) staining.
  2. Immunohistochemical staining using a panel of markers (CD15, CD30, CD3, CD20, and BCL2).
  3. CCL3 gene expression analysis.


Examination of hematoxylin and eosin and immunohistochemical sections

The 53 patients included nine patients with Hodgkin’s lymphoma and 44 patients with non-Hodgkin’s lymphoma. Of these, seven patients had follicular lymphoma, 16 had diffuse large cell lymphoma, five had small lymphocytic lymphoma, five had peripheral T-cell lymphoma, seven had anaplastic large cell lymphoma, and four had Burkitt’s lymphoma [Table 1].
Table 1: Clinical and laboratory data of the patients

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The study also included 20 samples of reactive lymph nodes (taken from 11 men and nine women, aged 15–70 years, with a mean age of 38.1±12.6 years). Informed consent was obtained from all participants.

Human MIP-1α level in serum was measured using a Human CCL3/MIP-1α Quantikine enzyme-linked immunosorbent assay kit (R&D systems, Minneapolis, Minnesota, USA) in all study groups.

Lymphoma patients underwent laboratory analysis [determination of complete blood count, erythrocyte sedimentation rate, serum glucose levels, and serum lactate dehydrogenase (LDH) levels; bone marrow examination; and kidney and liver function tests], radiological assessment (computed tomography of the neck, thorax, abdomen, and pelvis), and cardiac assessment (electrocardiography and echocardiography); thereafter, staging was performed according to the Ann Arbor staging system, and the risk factors were evaluated according to the International Prognostic Index (IPI) 16. Accordingly, standard treatment protocols and follow-up schedules based on published guidelines were selected 17.

CCL3 gene expression analysis

Total RNA was extracted from the paraffin-embedded tissue according to the Qiagen Protocol (QIAGEN GmbH, Hilden, Germany) for isolation of total RNA. The amount of isolated total RNA was estimated quantitatively by spectrophotometric measurement and qualitatively by electrophoresis. The eluted RNA was subjected to reverse transcription by the reverse transcriptase enzyme for conversion into DNA.

A random hexamer from the high-capacity cDNA kit (Applied Biosystems, Foster City, California, USA) was added to RNA samples for reverse transcription using the MultiScribe (Applied Biosystems) reverse transcriptase enzyme. The cDNA is used as a template to amplify the studied genes and the cDNA normalized using glyceraldehydes-3-phosphate dehydrogenase as housekeeping gene. The assay identification numbers of target and housekeeping genes are as follows: CCL3 (Assay ID # Hs00234142_m1) and GAPDH gene (Assay ID # Hs99999905_m1) using the primers and Probes assays listed in TaqMan Gene expression assays from Applied Biosystems.

DNA amplification of the CCL3 gene was carried out by real time PCR using the real time PCR Step One instrument and software (Applied Biosystems). This included an initial denaturation step for 10 min at 95°C, followed by 40 cycles of annealing and elongation for 15 s at 95°C and 1 min at 60°C, respectively.

Instrument raw data (fluorescence) for all the samples were converted into threshold cycles (Ct) using SDS 1.2 software (Applied Biosystems). Ct values were then imported onto an Excel worksheet for relative quantification (RQ).

For RQ calculation, the geometrical mean of GAPDH was used as a normalization factor.

Calculation of the results was performed by application of the comparative Ct method for RQ, 2-ΔΔCt



The difference in threshold cycles for target and reference genes is as follows:



where ΔCt,calibrator is the mean value for a healthy control human.



The fold change, defined as the ratio between the averaged normalized expression levels of targets in neoplastic and corresponding non-neoplastic samples, was calculated. Normalized RQ values were log 2 transformed for statistical analysis.

The relative expression level of CCL3 mRNA was found to be significantly upregulated, up to 10-fold in lymphoma patients compared with reactive lymphadenopathy patients.

The CCL3-normalized mRNA level in lymphoma patients compared with reactive lymphadenopathy patients was 11.5 (0.8–13.4), whereas in healthy controls it was 1.13 (0.5–3.8).

Statistical analysis

Statistical presentation and analysis of data in the present study was carried out; continuous data were expressed as mean±SD, whereas categorical data were expressed as number and percentage. Categorical data were compared using the χ2-test. Continuous data between two groups were compared using Student’s t-test. Different parameters were compared using Pearson’s correlation test. To measure the sensitivity, specificity, positive predictive value, and negative predictive value of serum CCL3, a receiver operating characteristic curve was generated. Associations between clinical and laboratory variables and the serum CCL3 level and CCL3 expression were studied using multiple linear regression analysis. Response criteria and overall survival rates are reported according to published guidelines 18. The survival charts were depicted on Kaplan–Meier plots. The expression level of the CCL3 gene in lymphoma samples was categorized as high or low on the basis of whether the target level was above or below the median expression value, respectively. Statistical significance was defined as a P-value of less than 0.05. Analyses were carried out using the SPSS program, version 17 (SPSS Inc., Chicago, Illinois, USA) and the GraphPad Prism software (GraphPad Prism Software Inc., San Diego, California, USA).


  Results Top


The clinical and laboratory data of the patients are presented in [Table 1].

There was a significant increase in the serum level of CCL3 in lymphoma patients (range 30–120 pg/ml, mean 72.5±24.3 pg/ml) compared with patients with a reactive lymph node (range 20–60 pg/ml, mean 31.5±12.6 pg/ml). Receiver–operator characteristic curve analysis showed a sensitivity of 81.1%, specificity of 95.5%, and accuracy of 94%, with a cutoff value greater than 55 pg/ml.

There was a significant increase in the serum CCL3 level with an increased degree of IPI [Table 2].
Table 2: Relationship between serum CCL3 level (pg/ml) and the International Prognostic Index

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There was a positive correlation between serum CCL3 and LDH levels, whereas there was a negative correlation between the serum CCL3 level and time of follow-up (months) in lymphoma patients [Figure 1] and [Figure 2].
Figure 1: Correlation between serum CCL3 (pg/ml) and LDH level (U/l) in lymphoma patients. LDH, lactate dehydrogenase.

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Figure 2: Correlation between the serum CCL3 level (pg/ml) and time of follow-up (months) in lymphoma patients.

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There were significant relations between treatment response and age, the LDH level, and the serum CCL3 level [Table 3].
Table 3: Relationship between treatment response and some parameters

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CCL3 mRNA was expressed in seven (35%) patients and absent in 13 (65%) patients with reactive lymph nodes, whereas it was expressed in 48 (90.5%) patients and absent in five (9.4%) patients with lymphoma.

The Kaplan–Meier curve showed that CCL3 mRNA expression was associated with a decreased survival rate in lymphoma patients [Figure 3].
Figure 3: Kaplan–Meier curve of CCL3 mRNA expression in lymphoma patients.

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There were significant relationships between CCL3 mRNA expression and both treatment response and IPI, whereas there was no relationship with mortality or relapse [Table 4].
Table 4: Relationship between CCL3 mRNA expression and different parameters

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The serum CCL3 level was only significantly associated with the survival rate in multivariate Cox analysis (P<0.001*; [Table 5]; however, in univariate analysis, CCL3 expression was significant [Figure 3].
Table 5: Multivariate regression analysis between survival rate and different parameters

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  Discussion Top


Lymphomas are malignant lymphoproliferative disorders. They have a wide range of histological subtypes, clinical presentations, and immunophenotypic, cytogenetic, and molecular features. This heterogeneity could affect optimal treatment. The accurate classification of patients should be dependent on reliable prognostic markers to obtain effective treatment.

Chemokines (chemotactic cytokines) are a large family of signaling molecules that are implicated in inflammatory and immune responses. They are also involved in tumor growth regulation and metastasis 19–21.

CCL3 is a member of the CC chemokine subfamily. It plays a role in the regulation of the activity of monocytes/macrophages by binding to CCR1 and CCR5 receptors, leading to the tumoricidal activity of these cells 22–23. CCR1–CCL3 is expressed in many tumors (hepatocellular carcinomas) and has a role in their progression 24.

Nakashima et al. 25 found that tumor cells transfixed with the CCL3 gene have reduced tumorigenicity and are able to induce antitumor activity. Vallet and Anderson 26 reported that CCL3–CCR1 had a main role in the pathogenesis of multiple myeloma and induction of osteolytic lesions.

Xu et al. 27 found that the serum CCL3 level was correlated with the grading of invasive gliomas and has a role in tumor proliferation and migration. They also found that it could be used as a prognostic marker for the tumor.

Terpos et al. 28 found that the serum CCL3 level was increased in patients with Waldenström’s macroglobulinemia (WM). CCL3 is released from the WM cells, thus, it can be used as a potential target for WM treatment.

In the present study, there was a significant increase in the serum CCL3 level in lymphoma patients, with a cutoff value of greater than 55 pg/ml. This increase was associated with the increased degree of IPI. There was a negative correlation between the serum CCL3 level and time of follow-up. There were significant relations between treatment response and age, the LDH level, and the serum CCL3 level. The serum CCL3 level was significantly associated only with survival rate in multivariate Cox analysis.

Hamed and Zaki found that the serum CCL3 level was increased in lymphoma patients, and there was a significant correlation between the serum CCL3 level and the overall survival of patients. Patients with a higher CCL3 level showed an increased percentage of deaths and relapse compared with patients with a normal serum CCL3 level 9.

Lossos et al. 29 identified serum CCL3 level as an independent predictor of survival in patients with diffuse large B-cell lymphoma.

Yan et al. 30 found that the serum CCL3 level correlated with survival and time-to-first treatment and was the most effective in distinguishing patients with chronic lymphocytic leukemia (CLL) from healthy individuals.

Sivina et al. 31 reported that plasma CCL3 level is an independent prognostic marker in CLL and should be used in risk assessment of patients with CLL.

CCL3 was expressed in 48 (90.5%) lymphoma patients and was absent in five (9.4%). The Kaplan–Meier curve showed that CCL3 expression was associated with decreased survival rate in lymphoma patients.

There were significant relationships between CCL3 expression and both treatment response and IPI, whereas there was no relationship with mortality or relapse because the size of the study group was small.

Hattori et al. 32 found that the serum level and expression of CCL3 were increased in patients with mantle cell lymphoma. CCL3 was an important factor in the development of both hypercalcemia and an aggressive phenotype in some types of B-cell lymphoma.

Mazur et al. 33 confirmed the higher CCL3 expression in lymphoma patients compared with patients with reactive lymphadenopathy.

Franke et al. 34 found that anti-CD20 treatment could be effective for lymphomas, as demonstrated through in-vitro studies. CD20 may play a role in breakpoint cluster region signaling by stimulation of CCL3 and CCL4 genes.


  Conclusion Top


The serum level or mRNA expression of CCL3 could be a valuable prognostic marker and may provide insight into creating a new therapeutic modality.[34]

 
  References Top

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32.Hattori N, Nakamaki T, Ariizumi H, Homma M, Yamochi-Onizuka T, Ota H, Tomoyasu S. Over-expression of CCL3 MIP-1alpha in a blastoid mantle cell lymphoma with hypercalcemia. Eur J Haematol. 2010;84:448–452  Back to cited text no. 32
    
33.Mazur G, Jaskuła E, Kryczek I, Dłubek D, Butrym A, Wróbel T, et al. Proinflammatory chemokine gene expression influences survival of patients with non-Hodgkin's lymphoma. Folia Histochem Cytobiol. 2011;49:240–247  Back to cited text no. 33
    
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    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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