|Year : 2019 | Volume
| Issue : 2 | Page : 153-161
Immunohistochemical expression of CD4+ and CD8+ tumor-infiltrating lymphocytes in colorectal carcinoma
Wafaa E Abdelaal, Naglaa F Abbas, Sonia L El-Sharkawy, Noha A.H. Helmy, Manal A Badawi, Dalia M Abouelfadl, Noha N Yassen, Marwa E Shabana, Ahmed S.A. Soliman, Ola R Oda
Department of Pathology, National Research Centre, Cairo, Egypt
|Date of Submission||26-Aug-2019|
|Date of Decision||19-Sep-2019|
|Date of Acceptance||21-Sep-2019|
|Date of Web Publication||26-Dec-2019|
Manal A Badawi
MD of Pathology, Department of Pathology, Medical Research Division, NRC, Elbohooth Street, Dokki Cairo 12622
Source of Support: None, Conflict of Interest: None
Background/aim Colorectal cancer (CRC) is one of the most common causes of cancer-related deaths in the world. The potential influences of tumor-infiltrating lymphocytes (TILs) in various neoplasms including CRC have been reported to reflect the antitumor immunity of the host. The aim of this study was to evaluate the expression of CD4+ and CD8+ TILs in CRC and to correlate their expression with patients’ clinicopathological parameters.
Materials and methods Paraffin blocks and clinicopathological data of 130 patients with CRC were obtained from private laboratories. The density of CD4 and CD8 tumor TILs was assessed immunohistochemically and evaluated by image analysis in 130 specimens of CRC. The CD4+and CD8+ cell tumor infiltrate was classified into scanty, moderate, and abundant expression.
Results Of the 130 CRC cases, 68 (66.2%) were adenocarcinoma and 44 (33.8%) were mucinous carcinoma, 114 (87.7%) were of grade II and 16 (12.3%) were of tumor grade III. CD4+ T cells expression was significantly correlated with the histopathological grade and Duke’s classification. CD8+ T cells expression was significantly correlated with the Duke’s classification.
Conclusion Cancer colon progression is influenced by host immune response and the presence of TILs CD4+ and CD8+. Further studies are recommended to assess implication of these cells in CRC prognosis.
Keywords: CD4+cells, CD8+cells, colorectal cancer, immunohistochemistry
|How to cite this article:|
Abdelaal WE, Abbas NF, El-Sharkawy SL, Helmy NA, Badawi MA, Abouelfadl DM, Yassen NN, Shabana ME, Soliman AS, Oda OR. Immunohistochemical expression of CD4+ and CD8+ tumor-infiltrating lymphocytes in colorectal carcinoma. J Arab Soc Med Res 2019;14:153-61
|How to cite this URL:|
Abdelaal WE, Abbas NF, El-Sharkawy SL, Helmy NA, Badawi MA, Abouelfadl DM, Yassen NN, Shabana ME, Soliman AS, Oda OR. Immunohistochemical expression of CD4+ and CD8+ tumor-infiltrating lymphocytes in colorectal carcinoma. J Arab Soc Med Res [serial online] 2019 [cited 2020 Oct 31];14:153-61. Available from: http://www.new.asmr.eg.net/text.asp?2019/14/2/153/274041
| Introduction|| |
The characteristics of cancer cells as well as the tumor microenvironment have been proposed to influence the biologic behavior of the tumors. Tumor-infiltrating lymphocytes (TILs), the primary immune response against cancer, were shown to reflect the immunity of the host and reflect the therapeutic outcome in patients with various types of tumors including lung and breast cancer ,.
Nowadays, TILs have been reported recently to attract attention as new biomarkers of colorectal cancer (CRC), the third most frequently malignancy worldwide. The incidence rates of colon cancer are higher in developed countries than in developing one. However, the mortality rate is higher in developing countries ,. Approximately, one million new cases of CRC are diagnosed annually throughout the world, with more than half a million death per year .
Recently, during development of CRC, the role of immune system in recognizing altered cells and in underlying the mechanism capable of eliminating tumors even in advanced stage has been a growing research interest ,.
Generally, the immune system has evolved several mechanisms to protect the host against cancer. Each of them has to be undermined or evaded during cancer development to enable tumor outgrowth. Among these; CD4+ and CD8+ T lymphocytes contribute to tumor suppression .
CD8+ T lymphocytes are crucial component of tumor-specific immunity which attack tumor cells ,. However, the role of CD4+ T cells is more complicated, as CD4 helper T cells have a central role in maintaining anticancer immunity, whereas CD4 regulatory T cells (T regs) suppress anticancer immune response promoting tumor progression ,,.
Although there have now been many studies suggesting a positive prognostic effect of CD4 and CD8, this still need to be verified in more studies. So the objective of this study was to evaluate the immunoexpression of both CD4 and CD8 in CRC.
| Materials and methods|| |
Specimens and ethical approval
We retrospectively obtained paraffin blocks and reviewed the database of 130 cases that underwent curative surgery for CRC from private laboratories. The patients had no other cancers or diseases, such as acute infection or diabetes, and they had not received previous radiotherapy or chemotherapy at the time of the surgery. This study was approved by the ethics committee of National Research Centre (Approval No. 16/308).
The histopathological characteristics were reviewed. The histological diagnosis was established and verified by two pathologists. Clinicopathologic data were retrospectively collected from medical records and are summarized.
The paraffin blocks were cut by microtome at 4 µ thickness. Sections were mounted on the charged glass slides. The slides were incubated at 37°C overnight for accurate adhesion of the section of the slide. Deparaffinization, rehydration, and epitope retrieval were performed in Pt link retrieval system (Dako, Copenhagen, Denmark).
The staining steps and incubation times were preprogrammed into the autostainer Link software (Dakoomnis, Copenhagen, Denmark). Diluted primary antibodies against CD4 and CD8 (Thermo Fisher Scientific, Runcorn, Cheshire, UK) and horseradish peroxidase labeled secondary antibody (Thermo Fisher Scientific) were used. For negative control, the primary antibody was replaced by normal mouse serum and human normal tonsil was used as positive control. Diaminobenzidine was used for color development and hematoxylin as counterstain.
Image analysis immunoscoring
Immunostaining was visualized and photographed under a light microscope Olympus CX-41 with DP 12 Olympus digital camera (Olympus Optical Co. Ltd, Tokyo, Japan).
Quantitative evaluation was performed by screening the sections and selecting at least five different fields with the high density of TILs. The density of CD4+ T cells and CD8+ T cells was determined using the Leica Qwin 500 image analysis system (LEICA Imaging Systems Ltd, Cambridge, England) which consists of Leica DM-LB microscope with JVC color video camera attached to a computer system. We selected five tumor fields with the highest expression and assessed the area percentage of the positive cells at high magnification (×200). We detected the positively stained cells, which were masked automatically by a blue mask called binary image. We used the measure field software program that automatically measures the area percentage of the detected features in the binary image and then displays the results in a table form. The immunoscore for CD4+ cells and CD8+ cells was evaluated according to Cho et al.  into scanty, moderate, and abundant expression.
Differences between groups were analyzed using the χ2-test. P values of less than 0.05 were considered to indicate statistical significance.
| Results|| |
The study population included 130 patients with CRC; there were 56 (43.1%) males and 74 (56.9%) females. The age of the patients ranged from 22 to 82 years, 66 (50.8%) of them were less than 60 years and 64 (49.2%) were greater than or equal to 60 years. [Table 1] shows all the clinicopathological characteristics of the studied cases.
Immunoscoring of CD4+ cells
A total of 42 cases showed scanty CD4+ T cells, with mean area percentage of 0.026±0.021 and range from 0.001 to 0.069%; 44 cases showed moderate CD4+ T cells, with mean area percentage of 0.126±0.046 and range from 0.070 to 0.201%; and 44 cases showed abundant CD4+ T cells, with mean area percentage of 0.565±0.316 and range from 0.202 to 1.319% ([Figure 1], [Table 2]).
|Figure 1 Immunohistochemical staining of CD4+ T cells in colorectal carcinoma and their binary images by Leica Qwin 500 image analysis system. (A1 and 2) Scanty expression, (B1 and 2) moderate expression, and (C1 and 2) abundant expression (CD4 immunohistochemical stain, Scale Bar: 200 μm).|
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CD4+ T cells expression was nonsignificantly correlated with all clinicopathological parameters, except the histopathological grade and Duke’s classification, which were significantly correlated with CD4+ T cells expression ([Table 3]).
|Table 3 Correlation between CD4 expression and clinicopathological features|
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Immunoscoring of CD8+ cells
A total of 44 cases showed scanty CD8+ T cells, with mean area percentage of 0.531±0.267 and range from 0.039 to 0.0999%; 42 cases showed moderate CD8+ T cells, with mean area percentage of 1.488±0.323 and range from 1.000 to 1.999%; and 44 cases showed abundant CD8+ T cells, with mean area percentage of 3.638±1.665 and range from 2.000 to 8.321% ([Figure 2], [Table 2]).
|Figure 2 Immunohistochemical staining of CD8+ T cells in colorectal carcinoma and their binary images by Leica Qwin 500 image analysis system. (A1 and 2) Scanty expression, (B1 and 2) moderate expression, and (C1 and 2) abundant expression (CD8 immunohistochemical stain, Scale Bar: 200 μm).|
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CD8+ T cells expression was nonsignificantly correlated with all clinicopathological parameters, except the Duke’s classification, which was significantly correlated with CD8+ T cells expression ([Table 4]).
|Table 4 Correlation between CD8 expression and clinicopathological features|
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Correlation between CD4+ and CD8+ T cells expression
Fourteen cases showed scanty CD4+ T cells and CD8+ T cells infiltration of tumor tissue. Twelve cases showed scanty CD4+ T cells and moderate CD8+ T cells infiltration of tumor tissue. Sixteen cases showed scanty CD4+ T cells and abundant CD8+ T cells infiltration of tumor tissue. Twelve cases showed moderate CD4+ T cells and scanty CD8+ T cells infiltration of tumor tissue. Sixteen cases showed moderate CD4+ T cells and CD8+ T cells infiltration of tumor tissue. Sixteen cases showed moderate CD4+ T cells and abundant CD8+ T cells infiltration of tumor tissue. Eighteen cases showed abundant CD4+ T cells and scanty CD8+ T cells infiltration of tumor tissue. Fourteen cases showed abundant CD4+ T cells and moderate CD8+ T cells infiltration of tumor tissue. Twelve cases showed abundant CD4+ T cells and CD8+ T cells infiltration of tumor tissue. There was a nonsignificant correlation between CD4+ and CD8+ T cell expression ([Table 5]).
|Table 5 Correlation between CD4+ and CD8+ T-cells in patients with colorectal carcinoma|
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| Discussion|| |
CRC is the third most common cause of cancer deaths in females and the fourth cancer-related mortality in males . In the present study, immunohistochemistry was used to detect the expression of T-cell markers (CD4 and CD8) in 130 cases of CRC.
Previous studies showed that CD8+ T cells destroy large tumor masses in vivo and patients with abundant CD8+ T cells in either tumor nest or in the stroma had a better prognosis . On the contrary, CD4+ T cells help prime CD8+ T cells responses and are essential for the maintenance of CD8+ T cell function by secreting cytokines required for CD8+ cell growth and proliferation . However, CD4+ TILs were classified into more subsets and the function of each one differs regarding antitumor immunity. T helper 1 (Th1) cells are a subset that activates CD8+ T cells, so they were reported to enhance antitumor immunity . On the contrary, T helper 2 (Th 2) cells are another subset that seems to suppress antitumor immunity via the production of immunosuppressive cytokine interleukins-10 . T helper 17 (Th 17) cells is the third subset that is reported by some authors to facilitate antitumor immune response. However, other authors showed that Th 17 cells may increase tumor growth through neoangiogenesis of the tumor . Regulatory T cells (T reg) were shown to suppress antitumor immunity.
In the present study, we used quantitative evaluation of area percentage of the positive cells to determine the density of CD4+ and CD8+ T cells in cases of CRC by immunohistochemistry. The immunoscore for CD4+ cells and CD8+ cells was evaluated according to Cho et al.  into scanty, moderate and abundant expression.
Our results showed scanty CD4+ T cells in 42 cases, with mean area percentage of 0.026±0.021. However, 44 cases showed moderate expression with mean area percentage of 0.126±0.046, and 44 cases showed abundant expression of CD4+ cells with mean area percentage of 0.565±0.316 within the tumor tissue. On the other hand 44 cases showed scanty CD8+ T cells expression with mean area percentage of 0.531±0.267, 42 cases showed moderate expression with mean area percentage of 1.488±0.323 and 44 cases showed abundant CD8+ cells expression with mean area percentage of 3.638±1.665. These results were comparable with those of other studies; however, the methodology for evaluating the densities of TILs has not been standardized.
Yasuda et al.  showed that the number of CD4+ was greater than or equal to 78 in 17 of 40 cases of CRCs and less than 78 in 23 cases, whereas CD8+ density was greater than or equal to 54 in 17 cases and less than 54 in 23 cases. Chirica et al.  studied CD4+ lymphocytes present in cancer colon and compared their numbers with those in neighboring unaffected mucosa. They showed that the percentage of CD4+ was higher in the tumor compared with unaffected mucosa. In support with this finding, Jasim et al.  showed similar results.
CD8+ T cells are cytotoxic T cells that play a critical role in attacking tumor cells with evidence that their densities were associated with long-term survival in various malignant tumors ,. Furthermore, it was reported that CD8+ T lymphocytes density was associated with the responses to radiotherapy and chemotherapy ,.
Our study showed that CD4 and CD8 lymphocytes infiltrate within the tumor tissue in direct contact with tumor cells which are often referred to as TILs. According to Guidoboni et al. , the presence of CD8+ T cells in stromal and peritumoral tissue was not correlated with better prognosis, whereas CD8+ T cells infiltrated within cancer cell nests were associated with good survival, independently of pathologic stage.Our results showed that no significant correlation between CD4+ and CD8+ cell infiltration of the tumor tissue with any of clinicopathologic parameters, except for significant correlation between histopathologic grade and CD4+ lymphocyte infiltration. There was also significant correlation between CD4+ and CD8+ cell infiltration of the tumor tissue and dukes classification. Similar results were reported by Matsutani et al.  who showed no significant association of CD8+ T cells with any of the clinicopathological except significant association of the density of CD8+ T cells and lymph node metastasis.
It has been reported TILs have a crucial effect on the tumor progression and clinical outcome in various types of cancer such as colorectal, esophageal, non-small cell lung cancer, bladder cancer and melanoma ,,,,.
In conclusion, data accumulated support the hypothesis that cancer colon progress is influenced by the host immune response and the presence of TILs (CD4+ and CD8+). Further studies are recommended to assess implication of TILs in CRC prognosis.
The authors are deeply appreciable of Professor Dr Abdallah M. Khalil and Professor Dr Elia A. Ishak for their support and help in collection of specimens.
This study was funded by National Research Centre − Egypt through a project within the 11th project plan.
Financial support and sponsorship
This study was funded by National Research Centre −Egypt. Project title: Immunohistochemical and image morphometric studies of tumor microenvironment of colon cancer 2016 - 2019 No. 11010112.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]