Central European Journal of Medicine

Peripheral blood CD4+CRTH2+ cells in advanced stage non small cell lung cancer

* E-mail: bulentkaragoz1968@yahoo.com

Received 14 December 2008; Accepted 16 February 2009

Abstract: To investigate CD4+CRTH2+ cells in peripheral blood in advanced stage non small cell lung cancer (NSCLC) patients. Forty-six patients with advanced stage NSCLC, who are chemotherapy or radiotherapy naïve, and 17 healthy volunteers, were enrolled in this study. The study was performed using flow cytometry and a complete blood cell counter analyser. CD4+ T cell percentage, CD4/CD8 ratio, CRTH2+CD4+ cell percentages, counts, and mean fluorescein intensity (MFI) and hematological parameters were evaluated in both groups. A survival analysis was performed to compare the patients with high CD4+CRTH2+ cell percentage and those with low CD4+CRTH2+ percentage. CD4+ T cell percentage in total lymphocytes and the CD4/CD8 ratio were lower in the patient group than in the control group. The absolute CD8 T cell count was higher in the patient group than in the control group, whereas the total T cells was not different. The CRTH2+ cell percentage in CD4+ T cells ( 7.96% ± 6.21% vs 3.37% ± 3.55%; respectively; p: 0,001) and the absolute count of CRTH2+CD4+ cells ( 97 mm-3 ± 109 mm-3 vs 37 mm-3 ± 38 mm-3, respectively; p: 0,033) in the patient group were higher than in the control group, but CRTH2-PE MFI values were not different between groups. Cox regression analysis did not show that CRTH2+CD4+ cell count or percentage is an independent prognostic factor. The study found that CRTH2 expression of CD4+ T cells and CRTH2+CD4+ cell number are higher in the peripheral blood of NSCLC patients than in that of healthy subjects. Further studies that explore the biological significance of high CD4+CRTH2+ cells in lung cancer patients, should be pursued.

© Versita Sp. z o.o.

Keywords: Lung cancer • CRTH2 • Th2 cells • Immunity • Survival

1 GATA Haydarpasa Training Hospital Department of Medical Oncology, Haydarpasa, 34667 Istanbul, Turkey

2 GATA Haydarpasa Training Hospital Department of Hematology, Haydarpasa, 34667 Istanbul, Turkey

Bülent Karagöz1*, Oğuz Bilgi1, Emin Gökhan Kandemir1, Alev Akyol Erikçi2, Özkan Sayan2, Alpaslan Özgün1, Bahattin Yılmaz1, Orhan Türken1, Ahmet Alacacıoğlu1, Ahmet Öztürk2

Research Article

1. IntroductionCRTH2 (chemo-attractant receptor, homologous molecule expressed by Th2 cells) is one of two known receptors of prostaglandin D2 (Pg-D2). It is principally expressed in Th2 cells, and also by osteoclasts, basophils, eosinophils and some monocytes [1-5]. CRTH2 expressing CD4+ T cells exist in greater numbers in peripheral blood from patients with allergies. Leukocyte chemotaxis (particularly eosinophil) is stimulated via CRTH2 [1]. Eosinophilia and eosinophil tumour tissue infiltration are favorable prognostic factors in lung cancer as well as in several cancer types [6,7].

Recently, the role of CD4+ T cells in a tumour immunology setting has been investigated [6]. Among

CD4+ T cells, CD4+CD25+ regulatory T (Treg) cells and T-helper 1 (Th1) subsets have frequently been studied. Finally, it has become apparent that there is an important role for Th2 cells in anti-tumour immunity [6]. Consequently, CD4+CRTH2+ cells may be important in the immune response against lung cancer. However, CD4+CRTH2+ cells have not been evaluated in peripheral blood of cancer patients yet.

In this study, we investigated for the first time CRTH2 expression of CD4+ T cells in non-small cell lung cancer (NSCLC) patients.

Cent. Eur. J. Med. • 5(4) • 2010 • 431-436 DOI: 10.2478/s11536-009-0047-0

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2. Material and Methods

2.1. Patients and study designForty-six NSCLC patients were enrolled in this study (Patients Group). Eighteen patients were stage III (IIIb and unresectable IIIA) and 28 patients had metastases (stage IV). Seventeen healthy volunteers, age matched, were included as control (Control Group). None of the patients received radiotherapy, or chemotherapy before the study. Exclusion criteria were as follows: previous malignancies, concomitant chronic infectious disease including human immunodeficiency virus, tuberculosis, immunosuppressive medications, existence of allergy, metastases of bone or bone marrow and non-steroidal anti-inflamatory drugs used in the previous 15 days. The study was approved by the Institutional Review Board. Demographic and clinical data were assessed. The TNM criteria were used for lung cancer staging. Peripheral blood was collected before the time of treatment for flow cytometry and complete blood count (CBC). Tumor response was evaluated after 3 cycles of chemotherapy by the use of consistent imaging techniques (CT or MR). Assessment was performed using the World Health Organization (WHO) criteria. Chemotherapy responses were documented as: objective response rate (complete response and partial response), and clinical benefit (complete response plus partial response plus stable disease).

Hematologic and flow cytometry data for the patients group and the control group were compared. These types of data were also compared for stage III and stage IV patients, and for patients with clinical benefit and patients with progressive disease. Then the median level of CD4+CRTH2+ cell percentages in CD4+ lymphocytes in the patient group was presumed as the cut-off point. Patients with CD4+CRTH2+ cell percentage lower than this level were separated from those with higher percentage (“Low CRTH2 Group” and “High CRTH2 Group”). A survival analysis was performed to compare the Low CRTH2 Group and the High CRTH2 Group.

2.2. Flow cytometryThree-color flow cytometry analyses were performed using Becton Dickinson FACSCalibur, and then analyzed with the Cell Quest program. Peripheral blood samples were obtained from patients and control subjects and the flow cytometry analyses were carried out again.

2.2.1. Monoclonal AntibodiesPhycoerythrin-cyanine 5 (PC5) conjugated anti-CD3 (eBioscience Catalog No:15-0038), PC5 conjugated anti-CD8 (eBioscience Catalog No:15-0088); fluorescein

isothiocyanate (FITCH) conjugated anti-CD4 (Caltag Lab Catalog No: MHCD0401) and phycoerythrin (PE) conjugated anti-CRTH2 (Santa Cruz Biotechnology Catalog No: sc-39838) antibodies and isotype controls were used.

2.2.2. Cell Preparation and Surface StainingHuman peripheral blood mononuclear cells were isolated by Histopaque (Sigma Catalog No:1077) gradient centrifugation. 100-μL aliquots were transferred to polypropylene test tubes (12x75 mm; BD Bioscience Catalog No: 352052 ). The cells were washed twice in PBS, and then incubated with 20-μL congugated monoclonal antibodies or isotype controls. After washing twice, the cells were fixed with phosphate binding solution (PBS) then analyzed by flow cytometry with BD FACSCalibur.

2.2.3. AnalysisCD4+ T cells, CD8+ T cells and CD4/CD8 ratios were calculated using CD4/CD8 double staining. Triple staining with CD4/CRHT2/CD3 was used for other cell counts. The percentage of T cells was counted in a lymphocyte gated SS/CD3 histogram. The absolute cell counts were calculated using absolute lymphocyte count from CBC. Subsequently, analysis of CD4+CRTH2+ cells (count and mean fluorescein intensity = MFI) was performed in a CD4/CRTH2 histogram.

2.3. StatisticsSPSS software was used for statistical analysis. Unpaired samples T test was used to investigate demographic, hematologic and flow cytometric parameters between groups. Gender distrubution was compared between groups by Fisher’s Exact test. CRTH2+ cell percentage in CD4 T cells, age and stage were investigated in survival using Cox regression analysis.

3. Results

3.1. Patient characteristics and clinical dataThe characteristics of the individuals in the patient and control groups are shown in Table 1, and this includes disease stage, primary treatment disciplines, and pathology. Three of the 46 patients in the study were followed up with supportive care because of the existence of comorbid diseases or low performance status. Two patients received primary radiotherapy. Forty-one patients were treated with palliative chemotherapy. None of the patients received chemoradiotherapy or had surgery. The combinations of platinum with docetaxel,

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paclitaxel, or gemcitabine were used for chemotherapy. In 24 patients, clinical benefit was obtained (17 with objective response) and 17 patients had progressive disease after first line chemotherapy.

Haemoglobin and haematocrit values were lower in the patient group than in the control group. Other hematologic parameters were not different between groups (Table 2).

3.2. Analysis of flow cytometryThe CD4+ T cell percentage in total lymphocytes and the CD4/CD8 ratio were lower in the patient group than in the control group, and the absolute CD8 T cell count was higher in the patient group than in the control group (Table 3). The total T cell count and percentage were not different. The CRTH2+CD4+ cell percentage in CD4+ T cells and the absolute count of CRTH2+CD4+ cells in the patient group were higher than in the control group

(Figure 1), but CRTH2-PE MFI values were not different between groups (Table 3).

The CRTH2+CD4+ cell percentage in CD4+ T cells (7.68% ± 6.3% versus 8.1% ± 6.2%; p:0.81), the absolute count of CRTH2+CD4+ cells (99/mm3 ± 118/mm3 versus 94/mm3 ± 104/mm3; p:0.88) and the CRTH2 antibody MFI value of CRTH2+CD4+ cells (16.9 ± 9.1 versus 14.8 ± 11.3; p:0.52) were not different between stage III and IV NSCLC patients.

Among 41 patients who received chemotherapy, the CRTH2+CD4+ cell percentage in CD4+ T cells, the absolute count of CRTH2+CD4+ cells and the CRTH2 antibody MFI value of CRTH2+CD4+ cells were also not different between patients with clinical benefit and patients with progressive disease [(6.32% ± 6.14% vs 8.97% ± 5.96%; p:0.15), (81/mm3 ± 101/mm3 vs 108/mm3 ± 112/mm3; p:0.43) and (14 ± 10.2 vs 15.3 ± 9.7; p:0.68) respectively].

Patients Control p

Number 46 17

Sex Female 6 4 0.438

Male 40 13

Age Median 58 61.5 0.104

Minimum 35 38

Maximum 80 85

Histopathology Squamous cell carcinoma 19

Adenocarcinoma 11

Unclassified carcinoma 16

Stage Stage III 18

Stage IV 28

Primary treatment modality Primary chemotherapy 41

Primary radiotherapy 2

Supportive care 3

Primary chemoradiotherapy 0

Surgery 0

Table 1. Study population characteristics.

Control Group Patient Group p

White blood cells (/μL) 7894 ± 1395 9517 ± 2969 0,005

Lymphocytes (%) 31.06 ± 6.91 31.24 ± 9.20 0,944

Absolute number of lymphocytes (/μL) 2411 ± 496 2886 ± 1063 0,082

Monocytes (%) 4.47 ± 2.10 4.59 ± 1.56 0,805

Absolute number of monocytes (/μL) 347 ± 155 435 ± 208 0,119

Granulocytes (%) 63.09 ± 8.12 58.47 ± 11.31 0,081

Absolute number of granulocytes (/μL) 5012 ± 1195 5713 ± 2508 0,141

Hemoglobin (g/dL) 14.1 ± 0.9 12.7 ± 2.0 0,001

Hematocrit (%) 42.04 ± 3.02 37.46 ± 5.65 <0,001

Platelets (x1000/μL) 210 ± 20 226 ± 102 0,307

* Mean ± standard deviation

Table 2. Hematologic Parameters*.

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3.3. SurvivalFor this part of the study, the patients were classified into two groups: the “High CRTH2+CD4+ Cell Percentage Group”, and the “Low CRTH2+CD4+ Cell Percentage Group”, depending on whether their CRTH2+CD4+ cell percentage was higher or lower than the mean value of the CRTH2+CD4+ cell percentage (7.96 % for NSCLC patients). Cox regresion analysis showed that stage was prognostic factor (0.033) for survival but, age and CRTH2 status were not (Figure 2).

4. DiscussionWe investigated for the first time CRTH2 expression of CD4+ T cells in NSCLC patients. We found increased number and proportion of CD4+CRTH2+ cells in peripheral blood of advanced NSCLC compared to healthy controls and no correlation between concentation of CD4+CRTH2+ cells in peripheral blood and survival,

Figure 1. Identification of CRTH2 expressing cells by flow cytometry. First lymphocytes, and then CD3+CD4+ cells were gated. Thus CD4+CRTH2+ cells are calculated on the basis of CD4/CRTH2 histogram.

Figure 2. Survival curve of High CRTH2 and Low CRTH2 Groups in non-small cell lung cancer. Cox regresion analysis did not show that CRTH2 ekspression of CD4 + T cells associated with survival data.

Control Group Patient Group p

CD4 T lymphocytes in total lymphocytes (%) 45.76 ± 10.52 35.79 ± 15.29 0,016

Absolute number of CD4 T lymphocytes (/μL) 1088 ± 299 1073 ± 680 0,904

CD8 T lymphocytes in total lymphocytes (%) 27.31 ± 5.31 34.20 ± 13.72 0,006

Absolute number of CD8 T lymphocytes (/μL) 658 ± 204 984 ± 541 0,001

T lymphocytes in total lymphocytes (%) 73.07 ± 12.75 69.99 ± 14.33 0,439

Absolute number of T lymphocytes (/μL) 1747 ± 442 2057 ± 993 0,093

CD4/CD8 ratio 1.74 ± 0.57 1.33 ± 0.89 0,037

CRTH2 + cells in CD4 T lymphocytes (%) 3.37 ± 3.55 7.96 ± 6.21 0,001

Absolute number of CRTH2+CD4+ T lymphocytes (/μL) 37 ± 38 97 ± 109 0,033

Mean Fluorescein Intensity of CRTH2 in CRTH2+CD4+ cells 20.11 ± 11.42 15.65 ± 10.41 0.146

* Mean ± standard deviation

Table 3. Flow Cytometric Parameters*.

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or response of chemoterapy. This study has several limitations. CD4+CRTH2+ cells in tumor tissue were not evaluated. Periferal blood Th2 cells were not determinated by cytokine secretion pattern. However, this study is unique in that it showed CD4+CRTH2+ cells in cancer patients.

CRTH2 is principally expressed in Th2 cells, and also by osteoclasts, basophils, eosinophils and some monocytes. Th1 CD4+ cells do not express the CRTH2 receptor. Although not all CRTH2+ T lymphocytes secrets Th2 type cytokines, existence of this receptor may be used as a marker for Th2 cells, and CRTH2+ cells can be determined by flow cytometry [8]. In the literature, peripheral blood percentages of CRTH2+CD4+ cells in CD4+ T cells have been reported as 0.4-6.5% in healthy subjects [9,10]. We also found a similar percentage of CRTH2+ cells in healthy volunteers.

Increased CRTH2+CD4+ cells have been observed in allergic diseases [1-3]. However, the status of CRTH2+CD4+ in diseases other than allergies remains to be elucidated. In this study, we report for the first time that CRTH2+CD4+ cells are more numerous in peripheral blood from NSCLC patients. Both the percentage of CRTH2+CD4+ cells in CD4+ T cells and the absolute count of these cells are higher in NSCLC patients compared with healthy subjects but the higher percentage of CRTH2+CD4+ cells is not associated with survival data in NSCLC patients.

A study showed that CRTH2+CD4+ cells have characteristics of activated effector and memory cells [10]. This research and our own results lead us to think that CRTH2+CD4+ cells may play roles in anti-tumour immune response. A study of breast cancer has showed more numerous CRTH2+CD4+ cells in tumour microenvironment compared with healthy tissue [11]. Last study also suggests a putative anti-tumour effect of CRTH2+CD4+ cells.

The intensity of the CRTH2 receptor in CRTH2+CD4+ cells is also important in several situations. In a study, decrease of intensity of CRTH2 on septic shock has been found. This decrease was associated poor clinical outcome [12]. We also investigated the MFI of the CRTH2-PE antibody from NSCLC patients but no difference was found between patients and control subjects.

Not all CRTH2+CD4+ cells are Th2 cells. Other cells that express CRTH2 may also play a role in anti-tumour immune response in NSCLC patients. However, CRTH2+CD4+ cells extensively comprise Th2 cells. Therefore, the more numerous CRTH2+CD4+ cells demonstrated in our study may be attributed to more numerous Th2 cells although Th2 cytokines were not investigated. The antitumour effect of Th2 cells has often been shown. Firstly, studies showed that the transfusion of Th2 cells inhibited tumour growth [13-15]. Secondly, interleukin-4 (IL-4), a Th2 cytokine, has been found to cause tumour inhibition [6]. This effect is obtained via augmentation of eosinophil and macrophages [16,17], or the anti-angiogenic effect of IL-4 [18,19]. Finally, IL-13, another Th2 cytokine, also plays a role in anti-tumour immune response via chemotaxis of myeloid cells [20-21].

In conclusion, this study found that CRTH2 expression of CD4 + T cells is increased in NSCLC patient compared healthy subjects. The advanced studies which explore biological significance of high CD4+CRTH2+ cells in lung cancer patients, should be established.

AcknowledgmentThe authors would like to thank Dr. Gulderen Yanikkaya Demirel and Mr. Lyle McClure.

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