Computerised counting of tumour infiltrating lymphocytes in 90 breast cancer specimens

Abstract

Tumour infiltrating lymphocytes (TILs) implicated in immunologic cytotoxicity were evaluated by immunohistochemistry and digitally counted in serial sections from 90 breast cancers in order to assess their number, the relationships between them and to tumour histology. CD3⁺, CD4⁺, CD8⁺, CD20⁺, CD25⁺ and CD56⁺ lymphocytes were found in 58 (64.4%), 52 (57.7%), 50 (55.5%), 22 (24.4%), 11 (12.2%) and 21 (23.3%) tumours, respectively. There was no difference in the number of TILs between pure infiltrating ductal (NOS) and non-ductal carcinomas, and no relationship between TILs and histological grades was found. CD3⁺TILs directly correlated to age, while lymph node negative patients had tumours infiltrated by fewer CD4⁺TILs with respect to lymph node positive patients. In 25/90 patients, randomly chosen, the status of peripheral blood lymphocytes was evaluated but no differences with respect to the status found in healthy blood donors was obtained; nonetheless while in some patients CD8⁺TILs outnumbered CD4⁺TILs in situ, the CD4/CD8 ratio was normal in their peripheral blood. The results show a considerable diversity of TILs among breast tumours, their lack of relationship with the status of the peripheral blood cells, and their potential important relationship with age (CD3⁺) and lymph node status (CD4⁺).

Introduction

Different studies conducted over the past decades have shown that inflammation in the tumour is a common feature of many malignant neoplasms [3], [13], [21]; inflammation is associated with good prognosis [17], poor prognosis [15] or of no effect [1], [18]. In breast cancer the role of tumour infiltrating lymphocytes (TILs) remains controversial, but it is clear that the T cell response to tumour cells is complex in terms of the molecules recognised on the latter and the different types of T cells activated during the response. The mechanisms of T-cell-mediated tumour cell killing require T cytotoxic and T helper cells together with macrophages and tumour antigens. Another set of lymphocytes, the natural killer (NK) cells, seems to have tumoricidal activity that is not dependent on prior sensitisation. Other cells, the lymphokine activated killer (LAK) cells, join the mechanism of immunologic tumour cytotoxicity [9], [10], [16]. Patients receiving interleukin 2 (IL-2) treatment have been reported to show changes in proliferative responses to IL-2 [2], [19], and to induce effector cells able to directly destroy NK-resistant target cells [8], [10], [11].

Since there is no general agreement as to how different classes of TILs associate between themselves and with different histological types of carcinoma, we have used monoclonal antibodies to evaluate the presence of some of the lymphocytes implicated in immunologic cytotoxicity in 90 breast cancers. Moreover, we developed a computerised system of counting stained cells within the tumour in order to obtain comparisons and correlations between the different classes of TILs. In a subgroup of patients we also evaluated the lymphocyte status in the peripheral blood in order to compare it to that found in healthy blood donors.