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ORIGINAL ARTICLE
Year : 2013  |  Volume : 50  |  Issue : 3  |  Page : 239-244
 

Presence of CD3+ tumor infiltrating lymphocytes is significantly associated with good prognosis in infiltrating ductal carcinoma of breast


1 Department of Pathology, King George's Medical University UP, Lucknow, Uttar Pradesh, India
2 All India Institute of Medical Sciences, Bhopal, Madhya Pradesh, (Formerly Professor, Department of Surgery, King George's Medical University UP, Lucknow, Uttar Pradesh), India
3 Central Drug Research Institute, Lucknow, India
4 Department of Pathology, Era's Lucknow Medical College and Hospital, Lucknow, Uttar Pradesh, India

Date of Web Publication23-Sep-2013

Correspondence Address:
M M Goel
Department of Pathology, King George's Medical University UP, Lucknow, Uttar Pradesh
India
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Source of Support: Indian Council of Medical Research, New Delhi (No. 5/13/26/2007/NCD III), Conflict of Interest: None


DOI: 10.4103/0019-509X.118744

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 » Abstract 

Background: Aim of this study was to investigate the prognostic significance of CD3+ TILs in infiltrating ductal carcinoma (IDC) of the breast. Materials and Methods: Immuno-histochemistry was done with CD3 antibodies in tissue sections of 127 breast cancer patients, and CD3+ intra-tumoral and stromal TILs were counted in relation to clinico-pathological variables. Results: Intra-tumoral and stromal CD3+ TILs were significantly associated with positive lymph node status (P = 0.006, P = 0.043, respectively) without significant association with age, menopausal status, family history, and hormonal status. The higher CD3 intra-tumoral and stromal counts both showed significant association with good prognosis (P = 0.039, P = 0.044, respectively). The intra-tumoral count was higher than stromal count and was independently associated with disease-free survival in stage I and II cancer (P = 0.021). Conclusions: CD3+ TILs may serve as independent marker of good prognosis in IDC breast. The findings of this study need further validation on a larger sample size.


Keywords: Breast cancer, CD3, infiltrating ductal carcinoma, intra-tumoral T-cells, stromal T-cells, tumor infiltrating lymphocytes


How to cite this article:
Rathore A S, Kumar S, Konwar R, Srivastava A N, Makker A, Goel M M. Presence of CD3+ tumor infiltrating lymphocytes is significantly associated with good prognosis in infiltrating ductal carcinoma of breast. Indian J Cancer 2013;50:239-44

How to cite this URL:
Rathore A S, Kumar S, Konwar R, Srivastava A N, Makker A, Goel M M. Presence of CD3+ tumor infiltrating lymphocytes is significantly associated with good prognosis in infiltrating ductal carcinoma of breast. Indian J Cancer [serial online] 2013 [cited 2019 Jun 26];50:239-44. Available from: http://www.indianjcancer.com/text.asp?2013/50/3/239/118744



 » Introduction Top


Breast cancer is the most frequent cause of cancer death in women worldwide [1] and is second most frequent cancer in females in India. [2] The conventional prognostic indicators in breast cancer include tumor type, histological grading, clinical stage, lymph node, and hormone receptor status. However, the tumors in different individuals have different biologic behavior.

Tumor infiltrating lymphocytes (TILs) in human cancers have long been thought to play a key role in inhibiting tumor proliferation, metastasis and may be an independent prognostic factor. [3],[4],[5],[6],[7] The TILs in the tumor indicate an anti-tumor cellular immune response. [8],[9],[10],[11],[12] The quantity of infiltrating lymphocytes within the tumor cell nests have been reported to correlate with an improved prognosis in many tumor types. [13],[14],[15],[16],[17]

The CD3 antigen, a pan T cell marker, is a protein complex comprising the T cell receptor, expressed on all T cells. The cells expressing this marker constitute a mixture of T cells including both T helper, T regulatory, and cytotoxic T cells. Controversy still surrounds the role and prognostic significance of TILs within a tumor microenvironment. Many studies have shown that it is a sign of the host immune response to tumor antigen [5],[18],[19] and a reflection of the dynamic process of cancer immune-editing. [20]

The present study was done to evaluate CD3+ subset of TILs, their localization, distribution, and density in breast cancer patients and their role on survival. The findings were correlated with known epidemiological and biological prognostic markers including tumor size, grade, lymph node status, estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor (CerbB2) status.


 » Materials and Methods Top


A total of 127 histologically proven cases of breast cancer, recruited from the Department of Surgery, were included in this study after informed written consent and institutional ethical clearance. Demographic details, clinical history, complete general/ local examination, and epidemiological risk factors including family history, clinical stage, tumor grade, lymph node status, estrogen receptor, progesterone receptor, and Cerb B2 were recorded on a detailed proforma designed for the study.

All patients had pre-operative tissue diagnosis of breast cancer either by fine needle aspiration cytology and/or by core biopsy of the breast. All the patients underwent surgery with axillary lymph node dissection, and none of these patients had received pre-operative anti-tumor therapy. Histological grading was done by Bloom and Richardson scoring. No patient had evidence of an active infection or inflammatory disease. Detailed histopathological examination was done in the Department of Pathology.

Immuno-histochemistry was performed using primary antibodies to CD3 (Novacastra, Leica Biosystem Newcastle Ltd., UK), ER, PR, and CerbB2 (Biogenex Laboratories, Inc, CA, US). Sections were de-paraffinized in xylene followed by hydration in graded Ethanol. Secondary antibody kit used was Novolink Min Polymer Detection System (Novacastra, Leica Biosystem Newcastle Ltd., UK).

Formalin-fixed, paraffin-embedded tissues sections (3-4 μm thick) were taken on 3-aminopropyl triethoxysilane (APTS) coated glass slides. Sections were immersed in antigen retrieval solution (citrate buffer pH 6.0), and antigen retrieval was done in the antigen-retrieval system (Biogenex Laboratories, Inc, CA, US) at 100°C for 20 min. The sections were then brought to room temperature. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 5 min and non-specific binding sites were blocked with protein block for 5 min. Sections were covered with 50 μl of individual primary antibody, kept in moist chamber, and slides were incubated overnight at 4°C. Slides were then washed with tris buffer saline (TBS), followed by a 30 minutes incubation with post primary block at room temperature (RT). Sections were then washed again in TBS and incubated with secondary antibody for 30 min at RT. 3,3'-diamino-benzidine was used as chromogen for visualization of antigen antibody complex. Sections were counterstained with hematoxylin, dehydrated through graded ethanol, cleared in xylene, and mounted with DPX. Negative control slides omitting the primary antibody were included in all batches. Section from tonsillar tissue served as positive control for CD3+ cells.

Microscopic evaluation and scoring of immunostained CD3 positive TILs was done independently by two pathologists. CD3 positive TILs were counted in five best fields showing highest number of lymphocytes under high power at 40X magnification, and the counts were averaged. The sections where lymphocytic infiltration was confined only to occasional areas, the counts from five contiguous high power fields were averaged. Initially, CD3+ TILs count was recorded as: + (1-25 cells), ++ (26-50 cells), +++ (≥51 cells) in the tumor and in the stroma separately. For the purpose of analysis, CD3+ TILs up to 25 cells were considered as low CD3+ TILs count and above 25 cells i.e. ++, +++ were considered as high count.

The mean follow-up period was 42 months. Follow-up data were available for 99 patients. Details of clinical progress and survival were obtained from the hospital records. During the follow-up period, 31 developed metastases and 5 died. The overall survival (OS) time was calculated for each patient to the nearest month, taken from the time of presentation to the time of death or last recorded follow-up.

Statistical Analysis was done by statistical software Stata 11.2 version (Stata Corp LP, College Station, Texas, USA). The data were described as number and percentages or mean ± SD, as applicable. Chi-square test (χ2 ) was used to test the association between categorical variables. Fisher's Exact test was applied for lower cell values. The age was compared by Mann Whitney test because of non-normal distribution of data. Kaplan-Meier curve were drawn for disease-free survival. Cox proportional hazard model was used for prediction of median time to relapse. In all cases, P-value <0.05 was considered as significant.


 » Results Top


The clinico-pathological characteristics of the patients included in this study are summarized in [Table 1]. The median age of study population was 50 years (range 25 - 86). Out of 127 patients, 2 women had undergone hysterectomy, and the clinical status of 99 (77.95%) women was available. Twenty-eight patients (22.02%) were lost to follow up. This was dropped from survival analysis taking recurrence (20.47%) as an event, and deaths (3.93%) were included in this group.
Table 1: Clinico-pathological details with relapse and survival data of 127 breast cancer patients

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The scoring of intra-tumoral CD3+ T-cells (ITTC) and stromal T cells (STC) infiltration ([Figure 1]a and b, respectively) in relation to clinico-pathological variables is shown in [Table 2]. CD3+ TILs were detected in intra-tumoral [Figure 1]c and stromal [Figure 1]d areas of immunostained histological sections of all the breast carcinomas. These were seen in abundance within the tumor cell nests. Intra-tumoral CD3+ TILs were significantly associated with lymph node status (P = 0.006). Though higher number of ITTC and STC were observed in clinical stages III, IV and histological grades 3, 4 as compared to lower stage and grades, the difference was statistically insignificant. The association of ITTC was insignificant in relation to age, menopausal status, family history, and combination of ER, PR, CerbB2 [Table 2]. Similarly, the STC correlated significantly with lymph node status (P = 0.009), whereas age, menopausal status, family history, stage, grade, and combinations of ER, PR, CerbB2 did not show significant association with CD3+ TIL count [Table 2]. Although a statistical correlation was found among CD3+ T-cell distribution (ITTC and STC) and clinico-pathological variables, no clear differences were found between IITC and STC distribution pattern.
Figure1: Intra-tumoral and stromal TILs in IDC (a) Intra-tumoral area of IDC (H and E x100) (b) Stromal area of IDC (H and E x100) (c) CD3+ TILs in Intra-tumoral area of IDC (Immunostain x100) (d) CD3+ TILs in stromal area of IDC (Immunostain x100)

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Table 2: Intra-tumoral and stromal CD3+ counts in relation to clinico-pathological characteristics

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Follow up data for relapse and disease-free survival for 99 patients showed that 26 patients had recurrence of the cancer [Table 2]. Five patients died, and all deaths were cancer-related. In univariate survival analysis, patients with higher intra-tumoral and stromal CD3+ T cell infiltration showed a longer overall survival (P = 0.039) and (P = 0.044), respectively. Cox regression analysis was used to verify whether the investigated variables concerning CD3+ TILs were valid or not [Table 3], [Figure 2]. The positive impact of intra-tumoral and stromal tumor infiltrating lymphocytes on overall survival was P = 0.004 and P = 0.028, respectively, and stage I and II of cancer (P = 0.021) was independently associated with disease-free survival. Higher number of CD3+ TILs was observed to be associated with better outcome.
Figure 2: Kalplan-Meier survival curves for CD3+ T cell infiltrating lymphocytes in breast cancer (a) Intra-tumoral CD3+ T cell infiltration (b) Stromal CD3+ T cell infiltration (c) Disease free survival according to stage (d) Lymph node status and survival

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Table 3: Hazard ratio for events using a Cox regression model

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 » Discussion Top


The study was undertaken with the aim to examine whether the presence of CD3+ TIL density and their location in infiltrating ductal breast carcinoma would have any prognostic significance. In the present study, we observed significantly high CD3+ counts in relation to positive lymph node status. Association of CD3+ positive TILs with other factors like age, menstrual status, histological grade, and tumor stage was not significant. High grade, stage, and lymph node positivity are known poor prognostic factors in breast cancer. Similar findings have been reported for medullary carcinoma of breast by other workers. [21]

The CD3+ TIL density may reflect the status of ongoing immune response against tumor cells. [22] The presence of CD3+ TILs has been attributed to the positive outcome in several studies. [9],[10] The high counts of CD3+ TILs compared to low counts have been reported to be associated with better survival in stage IB cervical cancer. [11] In a recent study, the presence of intra-tumoral CD3+ TILs was associated with better survival in epithelial ovarian cancer. [18] Higher stage was significantly linked with recurrence/death in our study. However, this may not be true for all types of cancers that tumors with high TILs will have good prognosis. This may vary even within the tumor types of the same organ. In a recent study of invasive micropapillary carcinoma of breast, TILs were associated with increased lymph node metastasis and a poorer prognosis. The results suggested that effective immunity provided by TILs varies in different tumors, and the relative lack of tumor-killing cytotoxic TILs in invasive micropapillary carcinoma may explain, in part, the adverse association of TILs with the biological behavior of invasive micropapillary carcinoma of breast. [21]

TILs form an important aspect of the host defense against the expansion of the solid neoplasms. It is a general belief that recruitment and accumulation of high concentrations of lymphocytes in the tumor tissue represent an important local barrier to neoplastic dissemination. It has been proposed that the improved clinical outcome is due at least in part to the presence of prominent TILs in the tumor. [23],[24],[25],[26],[27] The high intra-tumoral and stromal TILs in our study predicted longer survival (P = 0.039 and 0.044, respectively), implying that CD3+ TILs may act as good prognostic marker in IDC. Lymph node metastasis was present in 58/127 cases (45.6%) in our study with more number of TILs within the tumor as compared to stroma.

The technique of TIL counts is an important aspect. We used IHC for CD3+ TIL quantification, which had the distinct advantage of morphologically observing the TILs in tumor and stromal areas. Flow cytometry can also be used for CD3+ TILs, but fresh tissue is required for that. Moreover, direct visualization into intra-tumoral and stromal TILs will not be possible with flow cytometry. Further, the size of the histological sections taken for IHC is also important if morphological observations are to be made separately within the tumor areas and the stromal component. We performed all IHCs on larger tissue sections obtained from resected specimens, not on core biopsies. In a study, CD3+ TILs were seen by IHC in tissue microarray (TMA) constructs in 157 cases of ovarian cancer and they concluded that TILs can influence its prognosis depending on the location of TILs intra-tumoral versus stromal. [28] Although they used IHC for TILs, the major limitation in their study was the size of the tissue sections that was comparatively very small to observe small tumoral and stromal areas.

Why tumors rich in TILs have significant differences in behavior and clinical course is unknown, although several mechanisms have been suggested. It has been suggested that the functional status of TILs are different in different tumors, which might be Important for tumor immunity. The presence of immune cells in the tumor microenvironment, however, is not always a reflection of good prognosis. It is thought that the type and subtypes of immune cells will determine whether such "inflammation" is good or bad in terms of controlling and modifying the growth of cancer cells. [29],[30],[31] This dogma is central to the current 'immune-editing' hypothesis supported by many groups of researchers of the immune-biology of cancer. [20] Weakness of the present study was that only CD 3+ TILs were studied and that too only in one histological subtype of breast carcinoma i.e. infiltrating ductal carcinoma. The idea in our study was to find out whether IHC with infiltrating CD3+ TILs could be used as an adjunct to breast carcinoma work up like we do ER, PR, and CerbB2 in all cases. This could be helpful in stratifying the patients into high-risk or low-risk category at the time of tissue diagnosis on resected specimen. This criterion could be used, especially in resource-poor countries where there are always some financial constraints for molecular or advanced tests.

To summarize, the present study suggests that detection of density and location of CD3+ TILs in IDC could be an independent prognostic indicator. A larger sample size and also the different subsets of immune TILs in various histological types of breast cancer need to be studied and correlated with their outcome. Further studies are also needed to understand the underlying molecular pathways responsible for the association between intra-tumoral TILs and favorable prognosis.

 
 » References Top

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