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Applied Immunohistochemistry & Molecular Morphology:
doi: 10.1097/PAI.0b013e31828df3c9
Research Articles

Analysis of CD8+ and CD4+ Cells in Oral Squamous Cell Carcinoma and Their Association With Lymph Node Metastasis and Histologic Grade of Malignancy

Santos Pereira, Joabe dos DDS, MSc, PhD; da Costa Miguel, Márcia Cristina DDS, MSc, PhD; Guedes Queiroz, Lélia Maria DDS, MSc, PhD; da Silveira, Éricka Janine Dantas DDS, MSc, PhD

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Department of Oral Pathology, Oral Pathology Post-Graduation Program, Federal University of Rio Grande do Norte, Natal, Rio Grande do Norte, Brazil

The authors declare no conflict of interest.

Reprints: Éricka Janine Dantas da Silveira, DDS, MSc, PhD, Departamento de Odontologia, Programa de Pós- Graduação em Patologia Oral, Universidade Federal do Rio Grande do Norte, Av. Senador Salgado Filho, 1787, Lagoa Nova, Natal/RN, CEP 59056-000, Brasil (e-mail: ericka_janine@yahoo.com.br).

Received January 19, 2013

Accepted February 18, 2013

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Abstract

This study aimed to analyze CD8+ and CD4+ cells in oral squamous cell carcinoma and to correlate it with prognostic indicators. The sample was composed of 50 cases. Clinical data and histologic grade of malignancy were obtained. Specimens were submitted to immunohistochemistry. Cells were counted in 10 fields at the tumor invasion front and expressed as median. CD8+ cells were more frequent in nonmetastatic lesions (18.5) and in low-grade specimens (18.2) (P<0.05). CD4+ cells were equally distributed in nonmetastatic and metastatic lesions (4.5). In addition, they were slightly more frequent in low-grade lesions (4.7). None of these correlations were significant (P>0.05). CD8+/CD4+ ratio was higher in cases without metastasis (3.57) and in low-grade lesions (3.62) (P>0.05). Probably, CD8+ cells are the most effective and important cells in the host immune responses against oral squamous cell carcinoma. In addition, CD4+ cells could indirectly influence the host protection through regulation of CD8+ recruitment and activation.

Oral squamous cell carcinoma (OSCC) is the sixth most common type of cancer and is a huge problem of public health worldwide; besides, it is associated with important mortality and morbidity.1–3 An occurrence of almost half million new cases and 200,000 deaths annually have been estimated.4 OSCC usually occurs in patients in sixth decade of life, with history of tobacco and alcohol consumption, commonly involving the tongue.5,6

Despite advances in surgical techniques and the use of adjuvant treatments, the affected patients have low 5-year survival rate due to late detection and ineffective therapies.7 Therefore, efforts have been made regarding the basic research of oral cancer, in attempt to identify reliable biological markers for the biological nature and aggressiveness of these cancers.8

Histologic features allow the prognostic evaluation of these neoplasms, through histologic grade and clinical staging according to the TNM system, with assessment of tumor size and lymph node involvement.5 In addition, immunohistochemistry is frequently used to improve the histologic diagnosis.9

The features of OSCC typically comprise a stromal mononuclear immune inflammatory cell reaction.10 This reaction represents the host immune response that probably prevents against malignant transformation and proliferation of the cells, although this process can be insufficient.10,11 Transformed cells continually sensitize the immunologic surveillance system that provides the first line of defense against the progression of some tumors. The stromal mononuclear cell infiltrates are composed mainly of T cells and have been associated with a more favorable prognosis in patients with head and neck squamous cells carcinomas.12 CD8 and CD4 T cells compose the adaptive immunity, which is characterized by an antigen-specific response mediated by effector cells and antibodies.13

The CD8 T cells are a subpopulation of cytotoxic lymphocytes and are most likely effector cells that promote an efficient antitumor immunity.14 These cells can participate in the immunologic surveillance, recognizing, and killing potentially malignant cells, which express peptides from oncogenic viral proteins or mutant cellular proteins, presented with class I MHC.15,16

The role of CD4 T lymphocytes, also called T-helper cells, in tumoral immunity is not completely clarified yet. Therefore, CD4 cells may play a central role in initiating and maintaining anticancer immune responses.17,18 CD8 T cells can become lethargic or be deleted in the absence of CD4 T cells. Moreover, CD4+ cells are needed to imprint CD8+ cells with the ability to develop into long-lived memory cells. In addition, CD4+ cells could inhibit tumor growth in the absence of CD8+ cells by lysing MHC class II positive tumor cells or by promoting the recruitment of other effector cells.19

Although CD8+ and CD4+ cells recognize already identified head and neck tumor antigens, studies examining the role of host immune responses in the course of head and neck cancers are scarce. Such studies could increase considerably the knowledge about the relationship between immunologic responses and the development of OSCC. This may contribute to improve the therapies against cancer and support the development of new treatments. Thus, this study aimed to analyze the immunoexpression of CD8+ and CD4+ cells in OSCC intratumoral specimens, correlating it with presence or absence of metastasis and with histologic grade of malignancy.

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MATERIALS AND METHODS

This study was approved by the Ethics Committee of the Federal University of Rio Grande do Norte, Brazil (protocol 47/06), and was conducted according to the Declaration of Helsinki. Informed written consent was obtained from all subjects.

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Clinical and Morphologic Analysis

Fifty cases of OSCC fixed in formaldehyde and embedded in paraffin were obtained from the archives of the Discipline of Oral Pathology, Department of Dentistry, Federal University of Rio Grande do Norte. The specimens were acquired from patients submitted to tumor resection without prior chemotherapy or radiotherapy. Clinical data including presence or absence of metastasis at the time of diagnosis were obtained from registry files.

For morphologic study, histologic sections (5 µm) were stained with hematoxylin/eosin. The histopathologic features were assessed for the histologic grade of malignancy as previously proposed.20 The degree of keratinization, cellular pleomorphism, invasion pattern, and intensity of the inflammatory infiltrate were analyzed at the tumor invasion front. Individual scores were obtained for each aspect and then a total score was acquired. Cases were classified as low-grade malignancy (total score ≤8) or high-grade malignancy (total score >8).

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Immunohistochemistry

Histologic sections (3-µm-thick) were obtained and mounted on glass slides with organosilane (3-aminopropyltriethoxy-silane; Sigma Chemical Co., St Louis, MO). Then, they were submitted to immunohistochemistry by streptavidin-biotin method. Antigen retrieval was performed with citrate, pH 6.0, in a Steamer for 30 minutes. The specimens were incubated with the following primary antibodies: anti-CD8 (Dako, Glostrup, Denmark), diluted 1:20 for 60 minutes and anti-CD4 (Novocastra Laboratories Ltd., Newcastle, UK), diluted 1:20 for 120 minutes. The slides were counterstained with Mayer hematoxylin and cover-slipped with Permount (Thermo Fisher Scientific, Waltham, MA). As positive control, normal human tonsilar tissues, from prophylactic cervical lymph node resection were used. As negative control, the primary antibody was substituted by bovine serum albumin.

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Assessment of Immunoexpression and Statistical Analysis

The immunohistochemical expression of CD8 and CD4 in OSCC intratumoral specimens was analyzed considering the amount of immunopositive cells. This evaluation was made twice at different times by a single examiner blinded to the patient diagnosis. Cells were counted under light microscopy in 10 random histologic fields at the tumor invasion front (neoplastic nests and peritumoral stroma), with a magnification of ×400, adapted from Reichert et al.21 Then, the median value of these fields was established for each case and this immunoexpression was correlated with the presence or absence of metastasis and with the histologic grade of malignancy.

Descriptive statistics, the Mann-Whitney U test, and the Spearman correlation test were performed for analysis of the results using the SPSS software, version 20.0 (Chicago, IL). A P value <0.05 was considered to indicate statistical significance.

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RESULTS

Fifty patients with OSCC were studied: 34 (68%) males and 16 (32%) females. The median age of sample was 63.5±14.5 years (range, 31 to 90 y). Metastasis was absent in most of the sample (n=37, 74%) and present in 13 cases (26%). According to the histologic grade of malignancy, 26 (52%) cases were classified as low grade and 24 (48%) as high grade (Table 1).

Table 1
Table 1
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All cases studied were positive for immunohistochemistry, although there were differences concerning the amount of immunostained cells. CD8+ cells were more frequent than CD4+ cells in all specimens examined (median=16.0 and 4.5, respectively). Data regarding cell counting are presented in Table 2.

Table 2
Table 2
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Immunoexpression of CD8+ cells was more frequent in cases with absence of metastasis (median=18.5) when related to cases with presence of metastasis (median=12.5). This evaluation disclosed a significant difference (P<0.05). This difference was not observed in CD4 analysis, which revealed the same median of positive cells (median=4.5) in cases with and without metastasis (P>0.05) (Table 3).

Table 3
Table 3
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With respect to the correlation between immunoexpression of CD8 and histologic grade of malignancy, low-grade lesions exhibited a higher median of positive cells (18.2) than high-grade lesions (12.7). This result revealed a significant difference (P<0.05) (Figs. 1A, B). For CD4, however, no statistical difference was disclosed (P>0.05). Although not significant, CD4+ cells were slightly more frequent in low-grade malignancy lesions (median=4.7) when compared with high-grade cases (median=4.2) (Table 3) (Figs. 1C, D).

Figure 1
Figure 1
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The CD8+/CD4+ ratio was higher in cases without metastasis (median=3.45) and in low-grade lesions (median=3.57) when compared with cases with metastasis (median=2.12) and high-grade lesions (median=2.87). Despite that, this statistical analysis was not significant (P>0.05) (Table 4).

Table 4
Table 4
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Statistical analysis using the Spearman correlation test revealed a weak positive correlation between CD8+ and CD4+ cells, although not significant (r=0.123; P=0.394).

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DISCUSSION

OSCC is one of the most common malignant neoplasms, accounting for approximately 95% of oral cancers worldwide. The occurrence rates, high incidence, low 5-year survival rate, and difficulties in treatment together make this disease a significant problem of public health in many countries.3 An evidence of host immune response against cancer cells is the presence of tumor-infiltrating lymphocytes (TILs) surrounding the malignant cells in these lesions. This antitumor immunity is represented by various subsets of lymphocytes, including CD8+ and CD4+ T cells, which can assist in its effectiveness.22 Thus, this study analyzed the presence of CD8+ and CD4+ cells in a case series of OSCC.

Concerning to immunohistochemistry, the present study observed a higher frequency of CD8+ cells when compared with CD4+. According to literature, the TILs are mainly composed of CD8+ cells.12,23 Host immune defense against malignant cells is based on the action of TILs, although their role is not completely clarified. In this respect, the establishment of cancer could be caused by a weak immunogenicity of the malignant cells or by immunosuppressive effects of these cells. Cancer cells could escape host immune surveillance through these mechanisms.12,23,24

The present study found a significant association between a higher amount of CD8+ cells and the absence of metastasis. In addition, a higher amount of CD8+ cells was related to low-grade lesions. Watanabe et al22 studied the presence of CD8+ cells in the neoplastic nests and stroma of OSCC lesions. They verified that the lower amount of CD8+ cells was related to a shorter survival time. In their study, a shorter survival time was related to an advanced tumor stage and increased tumor grade. The study of Maleki et al13 observed similar findings. Verastegui et al24 verified that numbers of CD8+ cells were lower in metastatic lymph nodes from head and neck squamous cell carcinomas when compared with healthy lymph nodes. Piersma et al25 observed a strong association between the higher amount of CD8+ cells in the tumors and the absence of metastasis in cervical cancer.

In contrast to our results, Zancope et al14 did not observed significant differences between the amount of CD8+ cells and metastasis or histologic grade. However, they observed an inverse correlation between CD8+ cells in the peritumoral region and cell mitosis frequency at the tumoral front. The authors stated that CD8+ cells were related to a better prognosis and longer survival, being considered the most effective cells in the protection against malignant neoplasms.

Indeed, Kumagai et al12 stated that the action of cytotoxic CD8+ cells in the immune responses could inhibit the aggressiveness of malignant cells. In this respect, some studies considered the increased amount of CD8+ cells as an independent favorable prognostic factor in different types of cancer.25–29 CD8+ cells can recognize and kill potentially malignant cells, which express peptides from oncogenic viral proteins or mutant cellular proteins.15,16,26 Thus, our findings suggest that CD8+ cells cytotoxicity could participate in the prevention of OSCC development and growth.

In this study, no significant differences were observed between the number of CD4+ cells and metastasis or histologic grade. However, CD4+ cells were slightly more frequent in low-grade lesions. Similarly, Piersma et al25 did not find associations between the number of CD4+ cells and lymph node metastases in patients with cervical cancer. Sato et al27 observed no correlation between CD4+ cells and improved survival in patients with ovarian cancer. Despite that, these authors emphasize the importance of CD4+ cells as regulators of the positive prognostic influence exhibited by CD8+ cells.

Differently, Cho et al26 observed a significant correlation between increasing numbers of CD8+ and CD4+ cells and a better prognosis in patients with esophageal squamous cell carcinoma. Other studies suggested that CD4+ cells are essential for initiation and maintenance of anticancer immune responses.15

CD4+ cells could destroy tumors through the recruitment and activation of effector cells such as CD8+ cytotoxic cells, B cells, macrophages, eosinophils, and natural killer cells.15,26 Cytokines secreted by CD4+ cells have essential roles in the protection against tumors. The INF-γ has antitumor and antiangiogenic properties,30 whereas interleukin-2 is necessary for the maintenance of CD8+ cells effector functions including their growth and proliferation.26

Indeed, some studies have emphasized the cooperative role of CD4+ cells in the tumor eradication by CD8+ cells.26,31 On the basis of these findings, our results seem to indicate that although not significantly associated with metastasis or histologic grade, CD4+ cells could indirectly influence the prognosis through their action on CD8+ cells.

The present study observed that CD8+/CD4+ ratio was higher in cases without metastasis and in low-grade lesions, although not statistically significant (P>0.05). Piersma et al25 observed a significant association between higher CD8+/CD4+ ratio and absence of lymph node metastases in cervical carcinomas, thus indicating a favorable prognosis. Besides, they observed that lower CD8+/CD4+ ratio was related to failure of CD8+ cells to infiltrate the tumors in patients with metastasis. Other studies in different lesions observed that higher CD8+/CD4+ ratios were associated with improved outcome.27,32 Together, these results emphasize the important relationship between CD8+ and CD4+ cells in the prognosis of these patients.

Our results disclosed a weak positive correlation between CD8+ and CD4+ cells, which was not significant (P>0.05). This finding suggests that CD8 and CD4 cells have different functions in antitumor immunity; nevertheless, both cells have essential roles in this response.15,26,27

In conclusion, the current study revealed a significant association between the higher amount of CD8+ cells and absence of metastasis, and between this amount and low-grade lesions. This association also was related to CD8+/CD4+ ratio. Probably, CD8+ cells are the most effective and important cells in the host immune responses against OSCC. CD4+ cells were not associated with metastasis or histologic grade of the lesions. Furthermore, a weak positive correlation between CD8+ and CD4+ cells was observed. Thus, CD4+ cells could indirectly influence the host protection through regulation of CD8+ recruitment and activation.

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Keywords:

oral squamous cell carcinoma; CD8+ cells; CD4+ cells; metastasis; histologic grade of malignancy

© 2014 Lippincott Williams & Wilkins, Inc.

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