Flow Cytometric Analysis of the Lymphocyte Populations in Patients with Breast Carcinoma

Whitford, Philippa (1991) Flow Cytometric Analysis of the Lymphocyte Populations in Patients with Breast Carcinoma. MD thesis, University of Glasgow.

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Abstract

Evidence of antigen recognition and an anti-tumour immune response has been sought in patients with breast carcinoma since a lymphocytic infiltrate was first noted in the primary tumours and found to correlate with an improved prognosis 384. A variety of methods have been used to study immune competence but, with greater understanding of lymphocyte function, a clearer picture can be obtained by analysing the phenotypic proportions present in any lymphocyte population. The activation status of lymphocytes can also be deduced from their expression of activation markers and receptors, while the presence of surface IgG on the membrane of B cells is indicative of a mature humoral response. The aims of this study were: 1) To seek evidence of antigen recognition and an anti-tumour immune response in patients with breast cancer. 2) To assess the contribution of the axillary lymph nodes to any humoral or cellular immune response and the extent to which any response remains loco-regional at the time of clinical presentation. In this study, fluorescent monoclonal antibodies were used to stain the phenotypic and activation markers and flow cytometry was used to analyse the lymphocytes harvested from the primary tumours of 31 patients, the axillary lymph nodes of 40 patients and the peripheral blood of 39 patient. These were analysed with regard to the proportions of CD4+ helper T cells, CD8+ suppressor/cytotoxic T cells and B cells. We also looked at the proportion of each cell type carrying the activation marker HLA DR, the class II major histocompatibility (MHC) antigen, and the receptors for interleukin 2 (IL-2) and transferrin (Trf). In the case of the B lymphocytes, we stained surface membrane IgG. These parameters were also studied on the lymph node and peripheral blood lymphocytes of 7 control subjects undergoing vascular surgery or organ donation. The tumour cells themselves were stained for the expression of the class I and class II MHC antigens and for membrane bound IgG. The phenotypic proportions and activation status of the tumour infiltrating lymphocytes (TILs) were correlated with the tumour cell expression of class I and class II MHC antigens and with the common prognostic indicators of tumour stage, histological tumour grade and oestrogen receptor status. The composition and activation status of the lymph node and peripheral blood lymphocytes were correlated with tumour stage. PRIMARY TUMOUR TILs were found in 85% of the primary breast tumours studied and, in 60%, the infiltrate was sufficient to allow analysis of both the phenotypic and activation markers. T cells made up the bulk of the infiltrate with few B cells. CD8+ suppressor/cytotoxic T cells predominated overall, although CD4+ helper T cells were in the majority in 7 tumours with a strong infiltrate. The size of the CD8+ T cell population increased with histological grade (p< 0.05) and tumour cell expression of the class I MHC complex (p< 0.01). A large proportion of the CD8+ T cells were found to carry HLA DR and again this proportion increased with histological grade (p=0.003) and the tumour cell expression of both the class I and class II MHC complexes (p< 0.001). These findings suggest that activated CD8+ suppressor/cytotoxic T cells are being attracted by surface antigen, on poorly differentiated tumours, when this is presented along with the class I MHC complex. This latter is necessary for antigen recognition by CD8+ T cells. A similar pattern was seen with the CD4+ helper T cells but to a slightly lesser degree. However, a higher proportion of the CD4+ T cells than CD8+ T cells bore the receptors for interleukin 2 (p<0.0001) and transferrin, suggesting there is greater turnover or expansion of this cell type. There appears therefore to be some evidence, within the primary tumour, of the recognition of antigen and a cellular immune response to it. AXILLARY LYMPH NODES Lymph nodes from breast cancer patients were found to be large and engorged, with higher cell counts than nodes from control subjects. The nodes from patients with breast cancer had a higher proportion of CD4+ helper T cells (p=0.003) with CD4+/CD8+ T cell ratios ranging as high as 16:1 and averaging 5:1 (p=0.02). In the cancer nodes, a higher percentage of the T cells bore HLA DR (p< 0.0001) and, in the case of the CD8+ T cells this correlated with tumour stage (p=0.02). This again reinforces the association of this marker with the direct interaction of CD8+ T cells with antigenic tumour cells, in this instance, within nodal metastases. The IL-2 receptor was present on a greater proportion of the lymph node lymphocytes (LNLs) in the breast cancer patients (p=0. 006) and, as in the TILs, this receptor was present on more CD4+ than CD8+ T cells. Although the transferrin receptor was found on more lymphocytes in the nodes of breast cancer patients this was not statistically significant. Almost double the number of B cells were found to carry surface IgG in the breast cancer nodes as in the control nodes (p=0.03). There is evidence of helper T cell expansion and a humoral immune response in the axillary lymph nodes of patients with breast cancer. The CD8+ T cells appear more activated in patients with nodal metastases. (Abstract shortened by ProQuest.).

Item Type: Thesis (MD)
Qualification Level: Doctoral
Keywords: Biochemistry, Immunology
Date of Award: 1991
Depositing User: Enlighten Team
Unique ID: glathesis:1991-78300
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 28 Feb 2020 12:09
Last Modified: 28 Feb 2020 12:09
URI: http://theses.gla.ac.uk/id/eprint/78300

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