The immunophenotype and activation status of the lymphocytic infiltrate in human breast cancers, the role of the major histocompatibility complex in cell-mediated immune mechanisms, and their association with prognostic indicators

doi:10.1016/S0039-6060(03)00292-7

Surgery

Volume 134, Issue 5, November 2003, Pages 827-834

https://doi.org/10.1016/S0039-6060(03)00292-7 Get rights and content

Abstract

Background

The aims of this study were to characterize, phenotypically, the immune infiltrate in human breast cancers, to assess the activation status of tumor-infiltrating lymphocytes (TIL), and to define the association of these findings with established prognostic indicators.

Methods

Immunohistochemistry was performed on frozen sections of 60 primary breast cancers by use of monoclonal antibodies to T lymphocytes (CD3), T-helper cells (CD4), cytotoxic T-cells (CD8), natural killer cells (CD56), interleukin-2 receptors (IL-2R), and major histocompatibility (MHC) class I antigen (HLA-ABC) and MHC class II antigen (HLA-DR).

Results

All tumors stained positive for CD3, CD4 and CD8, but with marked variation in the intensity of the infiltrate. In tumors with a moderate infiltrate of TIL, there was a trend toward a greater representation of T–helper cells. However, as the intensity of TIL increased, there was a decline in the proportion of T–helper cells and a concomitant rise in the relative proportion of cytotoxic T cells. There was a relative paucity of natural killer cells. A significant association was found between the intensity of TIL and the number of positive nodes (P = .02) and the intensity of the infiltrate of both T–helper cells and cytotoxic T cells with ER expression (P = .03 and .05, respectively). Most tumors stained positive for IL-2R. The expression of IL-2R was associated with the intensity of TIL (P<.0001), T–helper cells (P<.002), cytotoxic T cells (P = .01) and natural killer cells (P = 0.04), and also with the degree of lymph node positivity (P = .02) and histologic tumor grade (P = .05). MHC class II expression was variable, and a large proportion of the tumors showed limited expression in individual cancer cells. There was an association between the expression of HLA-DR in tumor cells and the activation status of TIL (P = .03).

Conclusion

An immune infiltrate is an invariable finding in breast cancers, and the intensity of the infiltrate is greater in node positive tumors. Additionally, TIL may well be activated, albeit partially, in most tumors, suggesting that cell-mediated immune mechanisms are functionally intact.

Section snippets

Patients and methods

Samples of tumor were collected from 60 consecutive patients with primary breast cancer after informed consent was obtained. They were all treated either by wide local excision of the tumor or total mastectomy. Additionally, all patients underwent an ipsilateral axillary node dissection in accordance with our management protocol.

A fresh tissue sample was retrieved from the central area of each tumor specimen and immediately snap-frozen in liquid nitrogen (within 20 minutes of excision). There

Patient and tumor characteristics

Background data of patients with regard to age, menopausal status, histologic type of tumor, tumor size, and lymph node, ER, PR and HER-2/neu status are summarized in Table II.

T lymphocytes (CD3)

None of tumors had a CD3 score of 0. All tumor sections examined consisted of areas of solid sheets of neoplastic cells and areas of stroma, consisting mainly of fibroblastic cells that lay between the islands of tumor. TIL were present predominantly within areas of stromal tissue, either as aggregates or in isolation,

Discussion

Adenocarcinomas of the breast behave clinically and epidemiologically in ways that suggest that host resistance factors are important for outcome in addition to grade and stage of malignancy. Immune reactivity to autologous tumors is indicated by the presence of lymphoid infiltration and regional lymph node changes; however, these changes predict favorable outcome only in non-metastatic disease.17., 18., 19., 20., 21., 22., 23., 24., 25., 26., 27. Success of immunotherapy in human breast cancer

References (38)

T.A. Waldmann
The interleukin-2 receptor
J Biol Chem
(1991)
S.L. Swain
Lymphokines and the immune response: the central role of interleukin-2
Curr Op Immun
(1991)
P. Whitford et al.
Flow cytometric analysis of tumor infiltrating lymphocyte activation and tumor cell MHC class I and II expression in breast cancer patients
Canc Lett
(1992)
J.W. Hadden
The immunology and immunotherapy of breast cancer: an update
Int J Immunopharmacol
(1999)
B.M. Vose et al.
Human tumor-infiltrating lymphocytes: a marker of host response
Sem Hemat
(1985)
A. Jarry et al.
Same peculiar subset of HML1+ lymphocytes present within normal intestinal epithelium is associated with tumoral epithelium of gastrointestinal carcinomas
Gut
(1988)
K.L. Cepek et al.
Integrin alpha E beta 7 mediates adhesion of T lymphocytes to epithelial cells
J Immun
(1993)
K.A. Smith
T-cell growth factor
Immun Rev
(1980)
T.A. Waldmann
The structure, function, and expression of interleukin-2 receptors on normal and malignant lymphocytes
Science
(1986)
A.K. Bhan et al.
Immunohistologic characterization of major histocompatibility antigens and inflammatory cellular infiltrate in human breast cancer
J Natl Canc Inst
(1983)

D.J. Rowe et al.

Characterization of breast cancer infiltrates using monoclonal antibodies to human leukocyte antigens

B J Cancer

(1984)

J. Hurlimann et al.

Mononuclear cells infiltrating human mammary carcinomas: immunohistochemical analysis with monoclonal antibodies

Int J Cancer

(1985)

T.L. Whiteside et al.

Clonal analysis and in situ characterization of lymphocytes infiltrating human breast carcinomas

Canc Immun Immunoth

(1986)

J.A. Zuk et al.

Immunohistochemical analysis of HLA antigens and mononuclear infiltrates of benign and malignant breast

J Path

(1987)

R. Bilik et al.

Characterization of T-lymphocyte subpopulations infiltrating primary breast cancer

Canc Immun Immunoth

(1989)

A. Naukkarinen et al.

Quantitative immunohistochemical analysis of mononuclear infiltrates in breast carcinomas—correlation with tumor differentiation

J Path

(1990)

M.M. Black

Immunopathology of breast cancer

Pathobiol Annu

(1977)

G. Tonini et al.

Adjuvant treatment of breast cancer: a pilot immunochemotherapy study with CMF, interleukin-2 and interferon alpha

Cancer Immunol Immunother

(1998)

A.K. Stewart et al.

Adenovector-mediated gene delivery of interleukin-2 in metastatic breast cancer and melanoma: results of a phase 1 clinical trial

Gene Ther

(1999)

Cited by (61)

Evaluation of T, B and natural killer lymphocyte in the cervical stroma of HIV-positive and negative patients with cervical intraepithelial neoplasia
2016, Immunology Letters
Cervical intraepithelial neoplasias (CIN) are closely associated with oncogenic subtypes of the human papillomavirus (HPV). In the presence of this virus, it is known that the activation or suppression of immune system is the key to the development, progression and/or regression of cervical lesions. Therefore, the objective of this study is to compare the local immune response among HIV-seropositive and seronegative patients with cervical intraepithelial neoplasia regarding the expression of T lymphocytes (CD3+, CD4+ and CD8+), B lymphocytes (CD20+) and natural killers cells (CD56+) in the cervical stroma. A cross-sectional study of paraffin blocks containing cervical tissue after conization by the Loop Electrosurgical Excision Procedure (LEEP) from 47 HIV-seropositive and 38 seronegative patients with CIN. Cervical stroma immunohistochemistry was performed in the CIN area. The Fisher’s exact test was used for the statistical analysis. When HIV-seropositive and seronegative women were compared, the seropositive women had a higher count of CD8+ T lymphocytes (52.1% versus 28.9%, P < 0.04). Considering CIN degree (CIN 1 and CIN 2/3), the HIV-seronegative patients with CIN 1 had a low count of CD20+B-lymphocytes (7.1%) in comparison with CIN 1 HIV seropositive and with CIN 2/3 HIV-seronegative patients, respectively 50% (P < 0.018) and 54.5% (P < 0.0048). The HIV infection and degree of CIN influenced the cytotoxic lymphocytes inducing an increase in the number of cells high count of CD20+ lymphocytes with CIN 1.
Establishment of a heterotypic 3D culture system to evaluate the interaction of T<inf>REG</inf> lymphocytes and NK cells with breast cancer
2015, Journal of Immunological Methods
Three-dimensional (3D) culture approaches to investigate breast tumour progression are yielding information more reminiscent of the in vivo microenvironment. We have established a 3D Matrigel system to determine the interactions of luminal phenotype MCF-7 cells and basal phenotype MDA-MB-231 cells with regulatory T lymphocytes and Natural Killer cells. Immune cells were isolated from peripheral blood using magnetic cell sorting and their phenotype validated using flow cytometry both before and after activation with IL-2 and phytohaemagglutinin. Following the establishment of the heterotypic culture system, tumour cells displayed morphologies and cell–cell associations distinct to that observed in 2D monolayer cultures, and associated with tissue remodelling and invasion processes. We found that the level of CCL4 secretion was influenced by breast cancer phenotype and immune stimulation. We further established that for RNA extraction, the use of proteinase K in conjunction with the Qiagen RNeasy Mini Kit and only off-column DNA digestion gave the best RNA yield, purity and integrity. We also investigated the efficacy of the culture system for immunolocalisation of the biomarkers oestrogen receptor-α and the glycoprotein mucin 1 in luminal phenotype breast cancer cells; and epidermal growth factor receptor in basal phenotype breast cancer cells, in formalin-fixed, paraffin-wax embedded cultures. The expression of these markers was shown to vary under immune mediation. We thus demonstrate the feasibility of using this co-culture system for downstream applications including cytokine analysis, immunolocalisation of tumour biomarkers on serial sections and RNA extraction in accordance with MIQE guidelines.
Inflammatory breast cancer: New factors contribute to disease etiology: A review
2014, Journal of Advanced Research
Inflammatory breast cancer (IBC) is a highly metastatic and fatal form of breast cancer. In fact, IBC is characterized by specific morphological, phenotypic, and biological properties that distinguish it from non-IBC. The aggressive behavior of IBC being more common among young women and the low survival rate alarmed researchers to explore the disease biology. Despite the basic and translational studies needed to understand IBC disease biology and identify specific biomarkers, studies are limited by few available IBC cell lines, experimental models, and paucity of patient samples. Above all, in the last decade, researchers were able to identify new factors that may play a crucial role in IBC progression. Among identified factors are cytokines, chemokines, growth factors, and proteases. In addition, viral infection was also suggested to participate in the etiology of IBC disease. In this review, we present novel factors suggested by different studies to contribute to the etiology of IBC and the proposed new therapeutic insights.
Cytokines secreted by macrophages isolated from tumor microenvironment of inflammatory breast cancer patients possess chemotactic properties
2014, International Journal of Biochemistry and Cell Biology
Citation Excerpt :
Dissemination of carcinoma cells can be regulated by cues from the inflammatory cells within the tumor microenvironment. Macrophages, which are the major inflammatory cells that infiltrate into breast tumors (Mukhtar et al., 2011; Pollard, 2008), contribute to high levels of growth factors, hormones, and cytokines (Aaltomaa et al., 1992; Georgiannos et al., 2003) and are designated as tumor associated macrophages (TAMs). Within the tumor microenvironment macrophages polarize into heterogeneous subpopulations depending on the type of external stimuli they receive (Cassetta et al., 2011).
Although there is a growing literature describing the role of macrophages in breast cancer, the role of macrophages in inflammatory breast cancer (IBC) is unclear. The aim of present study was to isolate and characterize tumor associated macrophages of IBC and non-IBC patients and define their role in IBC. Tumor infiltrating monocytes/macrophages (CD14+ and CD68+) were measured by immunohistochemistry using specific monoclonal antibodies. Blood drained from axillary vein tributaries was collected during breast cancer surgery and the percentage of CD14+ in the total isolated leukocytes was assessed by flow cytometric analysis. CD14+ cells were separated from total leukocytes by immuno-magnetic beads technique and were cultured overnight. Media conditioned by CD14+ were collected and subjected to cytokine profiling using cytokine antibody array. Wound healing and invasion assays were used to test whether cytokines highly secreted by tumor drained macrophages induce motility and invasion of breast cancer cells. We found that macrophages highly infiltrate into carcinoma tissues of IBC patients. In addition blood collected from axillary tributaries of IBC patients is highly enriched with CD14+ cells as compared to blood collected from non-IBC patients. Cytokine profiling of CD14+ cells isolated from IBC patients revealed a significant increase in secretion of tumor necrosis factor-α; monocyte chemoattractant protein-1/CC-chemokine ligand 2; interleukin-8 and interleukin-10 as compared to CD14+ cells isolated from non-IBC patients. Tumor necrosis factor-α, interleukin-8 and interleukin-10 significantly increased motility and invasion of IBC cells in vitro. In conclusion, macrophages isolated from the tumor microenvironment of IBC patients secrete chemotactic cytokines that may augment dissemination and metastasis of IBC carcinoma cells.
Tumor-associated stromal cells expressing e-prostanoid 2 or 3 receptors in prostate cancer: Correlation with tumor aggressiveness and outcome by angiogenesis and lymphangiogenesis
2013, Urology
To clarify the detailed pathologic roles of prostaglandin E₂ in prostate cancer tissues, the present study investigated the clinical significance and prognostic roles of the density of tumor-associated stromal cells expressing specific receptors for prostaglandin E₂, termed “E-prostanoid (EP)1-4 receptors (EP1R-4Rs).”
The expression of each receptor was immunohistochemically examined in 114 formalin-fixed biopsy specimens. Correlations with clinicopathologic features were investigated in these specimens. Angiogenesis and lymphangiogenesis were measured by the percentage of CD34-stained vessels (microvessel density) and D2-40-stained vessels (lymph vessel density). The relationships between the density of each EPR-stained cells and the microvessel density or lymph vessel density were evaluated in 62 prostate cancer tissues obtained by radical surgery for more detailed analysis in a wider area of prostate cancer tissue.
The density of tumor-associated cells with EP2R expression was positively associated with the N (P < .001) and M (P = .002) stages. Similarly, EP3R-positive stromal cell density was significantly associated with the N (P = .033) and M (P = .026) stages. The density of EP2R- and EP3R-stained cells correlated with the microvessel density (r = 0.42, P < .001) and lymph vessel density (r = 0.36, P = .012), respectively. A greater density of EP2R-stained cells was recognized as an independent predictor of progression (hazard ratio 7.26, P = .002) on multivariate analysis.
EP2R- and EP3R-stained cells might play important roles in tumor progression, angiogenesis, and lymphangiogenesis in prostate cancer. The density of EP2R-stained stromal cells could offer a useful predictor of biochemical recurrence after radical surgery.
Local cytokine profiles of patients with cervical intraepithelial and invasive neoplasia
2012, Human Immunology
Citation Excerpt :
Finally, the slides were stained with hematoxylin and mounted properly for read in the microscope. The measurements of slides were analyzed according to the technique described by Georgiannos et al. [18]. Briefly, in this the quantity of marked cells is graded as: 0 absence of cells; 1, rare cells; 2 moderate number of cells; 3 many cells.
Several studies have suggested that patients with cervical intraepithelial and invasive neoplasia have reduced levels of Th1 cytokines, and increased levels of Th2 cytokines. Thus, the aim of this study was to delineate the immunological profile associated with lesion progression. Biopsies were obtained from 28 patients with low grade cervical intraepithelial lesions (LSILs), 53 patients with high grade cervical intraepithelial lesions (HSILs), 25 patients with invasive cancer (CA), and 20 healthy controls. Levels of IFN-γ, TNF-α, IL-2, IL-4, IL-10, IL-12, TGF-β1 and TGF-β2 were then assayed by RT-PCR and ELISA for each biopsy sample. For LSILs, higher levels of Th1 cytokines were detected, while HSILs were associated with a Th2 cytokine profile. In contrast, CA tissues were associated with the strongest expression of a Treg cytokine profile. In conclusion the most important contribution of these work is identification of the Treg cytokine profile in HPV progression lesions and in combination, these results suggested that tumor progression is dependent on suppression of cellular immunity.

View all citing articles on Scopus

View full text

The immunophenotype and activation status of the lymphocytic infiltrate in human breast cancers, the role of the major histocompatibility complex in cell-mediated immune mechanisms, and their association with prognostic indicators

Abstract

Background

Methods

Results

Conclusion

Section snippets

Patients and methods

Patient and tumor characteristics

T lymphocytes (CD3)

Discussion

J Biol Chem

Curr Op Immun

Canc Lett

Int J Immunopharmacol

Human tumor-infiltrating lymphocytes: a marker of host response

Sem Hemat

Same peculiar subset of HML1+ lymphocytes present within normal intestinal epithelium is associated with tumoral epithelium of gastrointestinal carcinomas

Gut

Integrin alpha E beta 7 mediates adhesion of T lymphocytes to epithelial cells

J Immun

T-cell growth factor

Immun Rev

The structure, function, and expression of interleukin-2 receptors on normal and malignant lymphocytes

Science

Immunohistologic characterization of major histocompatibility antigens and inflammatory cellular infiltrate in human breast cancer

J Natl Canc Inst

Characterization of breast cancer infiltrates using monoclonal antibodies to human leukocyte antigens

B J Cancer

Mononuclear cells infiltrating human mammary carcinomas: immunohistochemical analysis with monoclonal antibodies

Int J Cancer

Clonal analysis and in situ characterization of lymphocytes infiltrating human breast carcinomas

Canc Immun Immunoth

Immunohistochemical analysis of HLA antigens and mononuclear infiltrates of benign and malignant breast

J Path

Characterization of T-lymphocyte subpopulations infiltrating primary breast cancer

Canc Immun Immunoth

Quantitative immunohistochemical analysis of mononuclear infiltrates in breast carcinomas—correlation with tumor differentiation

J Path

Immunopathology of breast cancer

Pathobiol Annu

Adjuvant treatment of breast cancer: a pilot immunochemotherapy study with CMF, interleukin-2 and interferon alpha

Cancer Immunol Immunother

Adenovector-mediated gene delivery of interleukin-2 in metastatic breast cancer and melanoma: results of a phase 1 clinical trial

Gene Ther