| Abstract|| |
Background: The role of the tumor-infiltrating lymphocytes in invasive breast cancer and its correlation with different prognostic variables were always a matter of controversy in the literature. Aim: To determine the relative density of T lymphocytes, CD4+ cells, CD8+ cells, and B lymphocytes in breast cancer and assess their relationships with clinicopathologic parameters. Materials and Methods: Paraffin-embedded tissue sections from 48 invasive ductal carcinomas and 30 benign breast lesions were examined by means of immunohistochemistry to demonstrate CD3+, CD4+, CD8+, and CD20+. The immunophenotyped cells were semi-quantitatively graded into: Absent, intermediate, and extensive. Results: All lymphocyte populations were significantly more expressed in breast carcinomas than in benign lesions (P = 0.0001 for CD3+ and CD4+, P = 0.001 for CD8+, and P = 0.002 for CD20+ cells). In breast carcinoma, B and T cells were co-expressed in 33 of 48 tumors (68.8%). However, T cells were the predominant immunophenotype being noted in 81% of tumors, compared to B cells which were expressed in 50% of tumors. T cells, CD4+ and CD8+ cells were directly associated with patient's age (P = 0.004, P = 0.001, and P = 0.01, respectively). Clinical stages III and IV showed a significantly higher density of T and CD4+ lymphocytes than stage II (P = 0.004 and P = 0.009, respectively). Also, T and CD4+ cells were directly related to the histologic grade (P = 0.004 and P = 0.001, respectively). On the contrary, B lymphocytes were not related to any of the above-mentioned parameters. Conclusion: Although B and T lymphocytes were co-expressed in breast cancer, T lymphocytes and their subpopulations seem to have the upper hand in predicting the biological behavior. They probably promote neoplastic progression rather than acting as an antitumor immune response.
Keywords: Breast cancer, CD3+, CD4+, CD8+, CD20+, lymphocytes
|How to cite this article:|
Helal TE, Ibrahim EA, Alloub AI. Immunohistochemical analysis of tumor-infiltrating lymphocytes in breast carcinoma: Relation to prognostic variables. Indian J Pathol Microbiol 2013;56:89-93
|How to cite this URL:|
Helal TE, Ibrahim EA, Alloub AI. Immunohistochemical analysis of tumor-infiltrating lymphocytes in breast carcinoma: Relation to prognostic variables. Indian J Pathol Microbiol [serial online] 2013 [cited 2019 Apr 25];56:89-93. Available from: http://www.ijpmonline.org/text.asp?2013/56/2/89/118676
| Introduction|| |
Tumor-infiltrating lymphocytes (TILs) have long been recognized in breast cancer. ,,, Nevertheless, the exact role of the lymphocyte infiltrate with respect to the disease outcome has not been clearly defined. It has been suggested that the presence of TILs is an indication of favorable prognosis. ,, Conversely, some reports claimed that T lymphocytes play a role in the spread of carcinoma to lymph nodes. , The relationship of B lymphocytes to the prognostic factors in breast cancer has been investigated in a few studies. ,,
The controversy around the precise role of TILs has produced two competing hypotheses: One is that lymphocytic infiltrate merely reflects nonspecific inflammatory reaction resulting from tumor-derived chemokines and cytokines, and the other is that they represent a specific immunologic reaction. 
On the other hand, there is a disagreement on the T-lymphocyte subpopulations in breast cancer. Although some investigators have shown that T lymphocytes are mostly CD4+ cells, ,,, other studies reported that CD8+ lymphocytes represent a high proportion of T lymphocytes. ,,
The aim of the present work was to demonstrate the relative densities of T lymphocytes, CD4+ cells, CD8+ cells, and B lymphocytes in a series of breast carcinoma. Furthermore, the relationship between the density of each cell immunophenotype and the clinicopathologic factors of prognostic significance was assessed.
| Materials and Methods|| |
Specimens and data collection
This retrospective study included 48 breast carcinoma archival tissue specimens obtained from female patients who underwent excision biopsy or mastectomy at Ain Shams University Hospitals, Cairo, Egypt, during the period from January 2010 to June 2011. The criteria for selection of the specimens were: No history of preoperative chemotherapy, radiotherapy, or endocrine therapy, and cases diagnosed as invasive duct carcinoma, not otherwise specified (NOS).
Representative paraffin blocks and hematoxylin and eosin (HandE)-stained sections were retrieved from the pathology departments at Ain Shams University Hospitals. The patients' files were reviewed to record the patient age and the clinical stage of the disease. The latter was reported according to the American Joint Committee of Cancer.  The patients were grouped into two age groups (≤40 years and >40 years).
In addition to the breast carcinoma specimens, 30 tissue samples from age-matched benign proliferative breast lesions were collected to compare the pattern of TILs in malignant and benign breast lesions.
All patient data were collected and tissue specimens were retrieved in an anonymous way; so no consent was needed from the patients. The research study was approved by the Research Ethical Committee of Ain Shams University, the rules of which were in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki.
Representative HandE-stained tissue sections from all 48 breast carcinomas were examined for histologic grading of tumors according to Bloom and Richardson,  which classified invasive duct carcinoma (NOS) into grade 1 (well differentiated), grade 2 (moderately differentiated), and grade 3 (poorly differentiated). Histologic sections of both carcinoma cases and benign breast lesions were examined for the density of TILs which were semi-quantitatively graded as mentioned subsequently.
Formalin-fixed, paraffin-embedded tissue sections from the 48 mammary carcinomas and 30 benign breast lesions were immunostained for CD3+ (T-lymphocyte marker), CD4+ (helper/inducer T-lymphocyte marker), CD8+ (suppressor/cytotoxic T-lymphocyte marker), and CD20+ (B-lymphocyte marker). All antibodies were mouse monoclonal antibodies and available ready to use (Dako, Glostrup, Denmark). The immunohistochemical technique was performed by applying the supersensitive avidin-biotin detection kit (Biogenex, Fresno, CA, USA) and following the technique of Hsu and Raine.  Microwave antigen retrieval in citrate buffer (pH 6.0) was performed. In parallel, positive controls (using normal human tonsil) and negative controls (by omitting the primary antibody) were performed to ensure the correct staining procedure.
Scoring of the lymphoid infiltrate
TILs were evaluated in H and E-stained and immunostained tissue sections for both mammary carcinomas and benign breast lesions by two pathologists who were unaware of the clinical data. Areas of highest lymphoid density were selected and necrotic areas were avoided. The density of the total lymphocytic infiltrate in H and E-stained sections and the density of each cell immunophenotype in the immunostained slides was semi-quantitatively graded according to Kreike et al.  into: Absent, minimal (less than 10 lymphocytes/high-power field 40×), moderate (lymphocytes easily identified but no large aggregates), and extensive (large aggregates of lymphocytes in more than 50% of the tumor). For statistical purposes, minimal and moderate grades were grouped as intermediate.
Data were analyzed with SPSS for Windows. The frequencies of the various types of TILs in breast carcinomas and benign lesions were compared by means of Fisher's exact test. The relationships between the density of the immunophenotyped cells and the prognostic variables were determined by applying Chi-square test. Correlations between the density of the total TILs and each of the lymphocyte subsets were determined by Pearson's correlation coefficient. The level of significance was set at <0.05.
| Results|| |
Patient and tumor characteristics
The patients with mammary carcinomas were aged between 30 and 75 years (mean = 49.4 ± 12.9). The clinical stages of the disease were stage II in 30 cases, stage III in 12 cases, and stage IV in 6 cases. Histologically, all tumors were invasive ductal carcinomas (NOS). The histologic grades were grade 2 in 12 tumors and grade 3 in 36 tumors.
Examination of HandE-stained sections revealed that Tumor-infiltrating lymphocytes (TILs) were evident in 39 of the 48 carcinomas (81.3%). The infiltrating lymphocytes were predominantly noted in the stroma surrounding and separating the tumor masses or at tumor edge. Semi-quantitative scoring of the density of TIL showed that it was absent in 9 tumors, intermediate in 33, and extensive in 6 tumors.
Immunophenotyping demonstrated that the majority of TILs were T lymphocytes which were present in all 39 carcinomas with TIL (81.3% of all carcinomas) [Figure 1]a. On the contrary, B lymphocytes were noted in 24 of the 39 tumors with TIL (50% of all carcinomas) [Figure 1]b. Co-expression of T and B lymphocytes was evident in 33 tumors (68.8%). T and B lymphocytes were usually seen in close proximity [Figure 1]. CD4+ cells were a constant component of T lymphocytes in all 39 tumors [Figure 1]c, while CD8+ cells were noted in 27 of the 39 tumors [Figure 1]d. Thus, the ratio of tumors revealed CD4+ to those with CD8+ cells was 1.34.
|Figure 1: Immunophenotyping of tumor-infiltrating lymphocytes in breast carcinoma, ×200. (a and b) are consecutive sections: (a) CD3+ cells, note that lymphocytes are present in the stroma around tumor mass; (b) CD20+ cells, note that B lymphocytes are in close proximity to Tlymphocytes. (c and d) are consecutive sections: tumor mass with stromal infiltration by CD4+ cells (c) and CD8+ cells (d)|
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The frequency of total TILs, T lymphocytes, CD4+ cells, CD8+ cells, and B lymphocytes was significantly higher in breast carcinomas than benign breast lesions [Table 1].
|Table 1: Comparison of the frequency of tumor-infiltrating lymphocytes in breast carcinomas (48 cases) and benign proliferative breast lesions (30 cases)|
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On the other hand, the density of TILs, T lymphocytes, CD4+ and CD8+ cells showed a significant direct association with patient age (P = 0.003, P = 0.004, P = 0.001, and P = 0.01, respectively) [Table 2].
|Table 2: Relationship between the density of tumor-infiltrating lymphocytes and patient age in 48 breast carcinomas|
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When TILs were analyzed according to the clinical stage of breast cancer, stages III and IV tumors showed significantly higher densities of total lymphocytes, T lymphocytes, and CD4+ lymphocytes than stage II tumors (P = 0.003, P = 0.004, and P = 0.009, respectively) [Table 3].
|Table 3: Relationship between the density of tumor-infiltrating lymphocytes and clinical stage of 48 breast carcinomas|
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Similarly, poorly differentiated carcinomas (grade 3) had significantly higher density of TILs, T lymphocytes, and CD4+ cells than better differentiated tumors (grade 2) (P = 0.003, P = 0.004, and P = 0.001, respectively) [Table 4].
|Table 4: Relationship between the density of tumor-infiltrating lymphocytes and histologic tumor grade of 48 breast carcinomas|
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On the contrary, B lymphocytes showed no significant association with any of the above-mentioned parameters.
Correlation between the lymphocyte immunophenotypes and the total TILs revealed a highly significant positive correlation between each lymphocyte population/subpopulation and the total TILs [Table 5].
|Table 5: Correlation between total lymphocytes and lymphocyte subpopulations in breast carcinomas|
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| Discussion|| |
TILs are now considered as a hallmark of cancer development.  Several studies have shown that the presence of lymphocytic infiltrate in cancer tissue indicates the participation of immune response in the control and elimination of tumor cells. ,, However, this classic view of the role of immune cells in cancer has been supplanted by a more complex view of lymphocytes having both pro- and anti-tumor properties. 
In the present study, we have noted that the breast carcinomas had a significantly higher frequency of TILs and their subsets than benign proliferative lesions. This observation which is consistent with previous reports ,, may be attributed to the increase in the load of antigens on the malignant mammary cells, such as p53, BRCA1, and BRCA2.  These antigens can induce local activation and proliferation of TILs.  Furthermore, breast cancer cells may produce cytokines which recruit more lymphocytes. 
TILs were demonstrated in 81% of our breast carcinomas. This percentage approximates that reported by An et al.,  but is higher than that reported by Marsigliante et al.  This is possibly due to inclusion of larger numbers of poorly differentiated carcinomas in our study than in the series of Marsigliante et al.  As we shall see subsequently, poorly differentiated carcinomas have a significantly higher frequency of TILs.
More importantly, immunophenotyping revealed that T and B lymphocytes were co-expressed in 70% of tumors, a result that is supported by others. ,,,, This suggests the participation of both cell-mediated and antibody-mediated immunity in the pathophysiology of breast cancer. However, we found that the predominant cell population was T lymphocytes. This finding was supported by several other studies. ,,,,,
One of the most controversial issues with respect to the immunophenotyping and TILs in breast cancer concerns the relative abundance of T-lymphocyte subpopulations. In the present study, CD4+ cells were more prevalent than CD8+ cells. Although these data agree with some authors' reports, ,,, the reverse was reported by others. ,, The most possible causes for these contradictory results are variations in patients' or tumor selection, the use of different techniques (flow cytometry vs. immunohistochemistry), and the use of different methods in interpretation of immunophenotyping (quantitative vs. semi-quantitative).
Analysis of the relationship between the density of TILs and patient age demonstrated that T lymphocytes and their subsets CD4+ and CD8+ cells were directly associated with patient age. Such a relationship has been investigated in a few studies with discordant results. Marsigliante et al.  found that only T cells were directly associated with the patient age, thus supporting, in part, our results. Conversely, Menard et al.  reported no significant difference among different age groups with respect to the frequency of TILs. More recently, Mahmoud et al.  showed that the number of CD8+ lymphocytes was weakly inversely correlated with the age of breast cancer patients.
When TILs were investigated according to the clinical stage of disease, stages III and IV had a significantly higher density of T lymphocytes and CD4+ cells than stage I. The association between T lymphocytes with advanced disease may be due to the ability of these cells to produce vascular endothelial growth factor and fibroblast growth factor which stimulate lymphangiogenesis and angiogenesis, and consequently promote dissemination of cancer cells.  It is worth mentioning that our finding does not support an earlier study  which revealed that the intensity of T lymphocytes was significantly lower in stage III than in stage I and II breast carcinomas. The authors claimed that T-cell infiltration did not represent tumor-induced immunosuppression, but an inherent weak host immune surveillance which contributed to tumor progression.
As previously described, ,, T lymphocytes and T-cell subpopulations were significantly associated with higher histologic grades. This result suggests that the degree of tumor differentiation may influence the degree of immune response of the patient.
In comparison to T lymphocytes and their subsets, B lymphocytes had no significant association with any of the clinicopathologic parameters. Such relationship was investigated by a few reports that yielded the same results. ,,
| Conclusion|| |
The present study suggested that although T and B lymphocytes were expressed in breast carcinoma, T lymphocytes and their subsets (especially CD4+ cells) predominated quantitatively and qualitatively. They seem to promote neoplastic progression rather than acting as a protective immune response against cancer. However, a study with a larger number of cases is recommended.
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Eman A. A. Ibrahim
Department of Pathology, Faculty of Medicine, Ain Shams University, Alabbassiya Square, Cairo
Source of Support: None, Conflict of Interest: The authors report no
confl icts of interest. The authors alone are responsible for the content
and writing of the paper. They did not receive any fi nancial support from
any organization to fulfi ll this research.
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]