The diagnostic value of selected immune parameters in peripheral blood of dogs with malignant mammary tumours – a preliminary study

Canine mammary tumours (CMTs) constitute the most frequent neoplasms in female dogs. The epidemiological, clinical, biological and genetic diversity of these tumours is paralleled by different disease outcomes and choices of appropriate treatment (4, 17). Given the prevalence of CMTs, there is a need to search constantly for more precise diagnostic and prognostic methods that make possible the selection of appropriate therapies. Understanding how the antitumour activity of the immune system in dogs with a particular type of mammary gland tumour differs from this activity in dogs with other types of mammary gland tumour will improve the prediction of the course of a disease and of the response to therapy in specific patients. Cytometric investigation of peripheral blood in dogs with these tumours may be a means of rapid detection of metastases or micrometastases in these animals in the future (25).

There are several studies that have reported changes to the immune status in canine cancers, but most of them are focused on leukocyte immunophenotyping inside the tumour milieu (4, 28). However, cancer is a systemic disease that induces changes to the immune system as a whole. Therefore, an improved understanding of tumour immunology must include not only the tumour microenvironment but also the systemic immune landscape. Peripheral immune system investigation may facilitate canine mammary tumour diagnosis and prognosis (28).

The main adaptive immune cells are T and B lymphocytes and they play key roles in the induction of immune responses against canine mammary tumours (4). The composition of these cell subpopulations varies depending on the tumour type, and different lymphocyte subsets may cause neoplasms to progress in different ways and fit different prognoses (10). However, the importance of the T-cell-to-B-cell ratio in mammary gland tumours in dogs is not yet clear (25).

The cancer immunosurveillance system involves T cells that identify and eliminate tumour cells, leading to tumour rejection (28). The lineage of canine T cells is defined by the CD3 protein complex as a salient feature; therefore, anti-CD3 antibodies can be used to identify T cell markers in dogs (23). Changes in CD3⁺ lymphocyte percentages have been detected in dogs with different types of cancers (5), which suggests that the CD3 antigen may constitute a potential marker in canine mammary tumour diagnostic methods. Besides CD3⁺ lymphocytes, CD8⁺ cytotoxic T (Tc) cells are crucial to anti-tumour immunity, and the prevalence of such cells in tumour infiltrate means that the reaction of the body against neoplastic cells is mainly cytotoxic (1). Changes in these cell percentages in peripheral blood have been proven in many types of human and canine cancers; therefore, CD8⁺ Tc lymphocytes may convey valuable information on the immune status of canine cancer patients and may be a potential diagnostic factor in canine mammary tumours (16). Although the majority of T cell-based cancer research focuses on the anti-tumour CD8⁺ Tc cell immune responses, recent studies have revealed that CD4⁺ T helper (Th) cells develop and sustain effective anti-tumour immunity, doing so through several different mechanisms (24). For example, CD4⁺ Th cells aid anti-tumour CD8⁺ Tc lymphocyte functioning, support CD21⁺ B cells in antibody production, and indirectly kill autologous tumour cells in a major histocompatibility complex class II–dependent mechanism (24). That is why changes in the cell percentage which comprise this subpopulation may be related to the stage of tumour progression.

The CD4⁺/CD8⁺ ratio gives the proportion of CD4⁺ Th cells to CD8⁺ Tc cells and can help predict the likely course of various diseases (12). Many studies have revealed that a decrease in the relative percentage of CD4⁺ lymphocytes together with an increase in the CD8⁺ lymphocyte percentage may be related to impaired immune responses (12, 28). This phenomenon is observed in aging and immune senescence, and may lead to increasing incidence and severity of cancer in older dogs (28). Moreover, many cancers are more frequent among human AIDS patients and their CD4⁺ lymphocyte number and CD4⁺/CD8⁺ ratio decrease significantly during the progression of the disease (22). This suggests that tumour development may be connected with changes in the numbers of specific T lymphocytes. Further, it has been noted that the CD4⁺/CD8⁺ ratio is decreased in many human and animal neoplasms, including canine cancers (6). According to Ostroumov et al. (15), CD4⁺ and CD8⁺ T lymphocyte interplay exerts a controlling influence on tumour growth. Therefore, this ratio can be considered a potential diagnostic and prognostic factor for canine mammary tumours.

The role of B lymphocytes in cancer progression is still not well established, particularly in canines. There are some articles linking B cells with immunosuppressive or regulatory functions in neoplastic processes (14, 29). In human breast tumours, regulatory B cells may provoke cancer metastasis (14). In other studies, B cells have been described to possess a protective rather than an immunosuppressive function (18). It is possible that this discrepancy is caused by the presence of B cell subsets with distinct phenotypes and different functions. As the processes of tumour eradication and tumour progression are related to the role of canine peripheral B lymphocytes, it is worth knowing whether the number of these cells changes in the course of the neoplastic process (21).

The present investigation aimed to assess the differences between the percentages of selected lymphocyte subsets in peripheral blood from healthy dogs and dogs with two different stages of malignant mammary tumour. In this study, single antibodies were used, which could become simple and relatively cheap diagnostic reagents if they fulfil their promise. The findings of this study concerning the quantity of essential circulating immune cell subtypes in mammary cancer may also improve the prospects of improving treatment strategies.

The gating strategy for this experiment is presented in Supplementary Fig.1. The cytograms of CD3 staining are presented in Supplementary Fig. 2. As shown in Fig. 1A, the mean CD3⁺ lymphocyte percentage in group M was significantly decreased compared to both group H and group WM (P-value < 0.05). However, there was no significant difference between the mean percentages in these latter two groups (40.39% and 35.48%, respectively).

Fig. 1.

Similarly to the CD3⁺ lymphocyte percentage results, those for CD21⁺ also did not differ statistically significantly between group H and group WM. However, a statistically significant increase was discovered in animals from group M over the control dogs’ percentage (P-value < 0.05), with mean values of 20.07% and 10.85%, respectively. A significant difference was also noted between the CD21⁺ lymphocyte percentages in the two groups of tumour-bearing dogs (P-value < 0.05) (Fig. 1B).

Figure 1C shows the CD3⁺/CD21⁺ cell ratio calculated for all groups. The ratio in group WM was significantly increased over that in group H. In contrast, the CD3⁺/CD21⁺ cell ratio was significantly decreased in patients with histological grade G2 tumours from that in group H (P-value < 0.05).

This was caused by the fall in CD3⁺ lymphocytes and the rise in CD21⁺ lymphocytes in group M. There was also a significant difference between group WM and group M.

Representative results of CD4 cytometric staining are given in Supplementary Figs 3A–C. The mean CD4⁺ lymphocyte numbers were compared statistically between all groups. There was no difference between the means of group H and group WM. However, statistically significant differences were observed between the former group and group M and between group WM and group M (P-value < 0.05). The number of CD4⁺ lymphocytes was lower in group M than in group H and group WM (Fig. 2A).

Fig. 2.

Representative results of CD8 cytometric staining are given in supplementary Figs 3 D–F. The mean CD8⁺ cell percentages were comparable between all groups and no statistically significant difference was observed (P-value < 0.05). The means were 18.36% for group H, 20.69% for group WM and 19.25% for group M. However, the variability of group M data was greater than that of group H (Fig. 2B and the standard deviation column in Table 2).

The CD4⁺/CD8⁺ ratio was significantly lower in group M compared to group H (Fig. 2C). This effect was produced mainly by a fall in CD4⁺ lymphocyte content, because the CD8⁺ lymphocyte percentage remained at a similar level in all groups.

Many studies suggest that tumours are able to exert systemic effects and influence peripheral blood leukocyte composition through tumour-derived cytokines and microvesicles to facilitate progression and metastasis (7, 10). There is growing evidence that tumour development, the recruitment of immune cells into the tumour site, and the presence of tumour-infiltrating leukocytes in tumour milieu are accompanied by changes in the numbers of specific types of leukocytes in peripheral blood (3, 10). The immunophenotyping of peripheral blood lymphocytes described in this article was predicated on the hypothesis that bitches with malignant mammary tumours exhibit an immune profile that is different from that of healthy dogs. Although there are some studies concerning leukocyte immunophenotyping in dogs with mammary tumours, some of the results are contrary; also, there are no standard values for leukocyte percentages in this disease. The innovation of this study lies in its attempt to evaluate single surface proteins to find prognostic and diagnostic markers.

In the present study we documented changes in the parameters of immune status in tumour-bearing dogs with malignant mammary tumours at different clinical stages (Table 2). There were no significant differences in the percentage of CD3⁺ T lymphocytes between the control group and the group of female dogs with early stage tumours. Contrastingly, in the group of dogs with metastasising tumours, a significant decrease in the percentage of CD3⁺ lymphocytes was noted. This is in line with the results obtained by Watabe et al. (28) and Garcia-Sancho et al. (5). The reason for the reduction in CD3⁺ cells is currently unknown, but may be that cytokines or other immunosuppressive factors secreted by the tumour downregulate the expression of the CD3 protein (28). Another hypothesis is that the decrease in the percentage of CD3⁺ lymphocytes in peripheral blood may be due to the infiltration of these lymphocytes into the tumour. A high percentage of CD3⁺ T lymphocytes has been observed in tumour microenvironment research (20). Given the noted significant difference between group WM and group M, CD3 receptor expression may be useful in differentiating metastasising and non-metastasising tumours and presumably in the prognosis of disease progression in bitches with malignant mammary tumours.

Our findings showed a significant rise in CD21⁺ peripheral blood lymphocyte percentages in the group of female dogs with metastasising tumours. This is contrary to the findings of Watabe et al. (28); however, their research included samples from dogs with various types of cancer, rather than only mammary tumours. In the study of Estrela-Lima et al. (4), the percentage of B lymphocytes was also significantly lower in dogs with cancer. This may be explained by the percentage of CD21⁺ lymphocytes possibly varying depending on the histopathological type of the tumour. The latest studies have proved that B cells can play a dual role in tumorigenesis. Regulatory B cells promote the development of cancer, while other types of B cell act to reduce cancer progression (8). Subsets of B cells may contribute to tumour growth because they promote angiogenesis and are found to be increased in cancer and chronic inflammation (26). The tumour environment is a location where B cells were found to exert immune-regulatory functions, secreting anti-inflammatory cytokines and thus inhibiting anti-tumour immunity (8). Estrela-Lima et al. (4) observed high levels of tumour-infiltrating B-cells in animals with worse prognoses (4). Additionally, the rise in the percentage of CD21⁺ lymphocytes may be evidence of the prevalence of the mechanisms of the Th2 humoral response over the Th1 response in bitches with mammary gland cancer at an advanced stage. The predominance of Th2 mechanisms is related to the downregulation of adaptive immunity, which is involved in an effective anti-tumour response (11). In addition, the prevalence of Th2 responses has been shown to be associated with the chronic inflammatory status of animals and tumour progression (30). From the results of the current research, it can be assumed that B cells contribute to tumour progression, as their increase is associated with the occurrence of metastases in female dogs with malignant mammary gland tumours.

Although there are some studies concerning peripheral lymphocyte phenotyping in tumour-bearing dogs (5, 28) and in bitches with mammary cancers (4, 13), there are few CD3⁺/CD21⁺ cell ratio data. The present study has shown that in the group of bitches with non-metastasising mammary tumours, both CD3⁺ T lymphocyte and CD21⁺ B lymphocyte percentages remained unchanged. That is why the CD3⁺/CD21⁺ ratio in the group without metastases was similar to the ratio in the control group. However, in the group with metastases the CD3⁺/CD21⁺ ratio was significantly decreased from the ratios in the control group and the group without metastases. This was due to a significant decrease in the percentage of CD3⁺ lymphocytes in combination with an increase in the percentage of CD21⁺ lymphocytes in bitches with metastatic tumours. Therefore, it can be concluded that a downward trend in the percentage of CD3⁺ T lymphocytes, especially in combination with an upward one in the percentage of CD21⁺ B lymphocytes, may be helpful in diagnosis and indicate the presence of metastases in female dogs with mammary gland cancers. The dominance of the Th2 humoral response and chronic inflammation in bitches with mammary tumours may be correlated with the advancement of the neoplastic disease and a poor prognosis (1, 5).

In the current research we observed that the percentage of CD4⁺ lymphocytes in the group of dogs with metastases was significantly lower than that in the group of dogs without metastases and the control group (P-value < 0.05). These findings are consistent with the studies of García-Sancho et al. (5) and Watabe et al. (28), which identified decreasing CD3⁺ and CD4⁺ cell numbers in dogs with different types of cancers. These results suggest that in bitches with malignant tumours at an advanced stage, the immune system is severely affected by the tumorigenic process.

Our findings indicate that there were no statistically significant differences in CD8⁺ lymphocyte percentages between the investigated groups (P-value < 0.05). These results are similar to those published by Mucha et al. (13). The results of other investigations are contrary to ours (5, 28), but those studies were conducted on groups of dogs with different types of tumour. It has been proved that the state of the immune system and the types of cells that are present in the bloodstream are related to the type of neoplasm (27). It is worth emphasising that we did not assess the differences in the activity of CD8⁺ lymphocytes between the groups of dogs, but only the differences in their percentages. It has been observed in some cancer studies that even if the number of CD8⁺ T cells remains unchanged, some of them may display features of dysfunction or exhaustion (9).

The CD4⁺/CD8⁺ ratios for the control group, the group with non-metastasising tumours and the group with metastasising tumours were 2.192, 1.967 and 1.361, respectively. The means of this ratio are usually between 1.7 and 2.8 in healthy dogs; the shortening of this ratio in the dogs with tumours may be evidence of immunosuppression (13). The CD4⁺/CD8⁺ ratio was decreased from the control group’s ratio in the group without metastases, albeit statistically insignificantly. A significant decrease was observed from the control group’s ratio to that of the group of bitches with metastases. Based on these results, it may be concluded that in dogs with malignant mammary tumours, the CD4⁺/CD8⁺ ratio decreases gradually with tumour progression. The noted significant shortening of the CD4⁺/CD8⁺ ratio in the group with metastases was due to a diminution of CD4⁺ cells, as the percentage of CD8⁺ cells was similar to that in the control group. A similar ratio change was also observed by Mucha et al. (13), in which metastasising bitches with mammary tumours had the lowest CD4⁺/CD8⁺ ratio. A low CD4⁺/CD8⁺ ratio is associated with immune senescence, activation and inflammation, which may all contribute to carcinogenesis (2). It has been proved that the CD4⁺/CD8⁺ ratio changes during the progression of many human and animal tumours, including those of dogs, and that it may be a potential diagnostic factor (2, 6). With respect to the lymphocyte dysfunction that seems to be present in cancer patients, a tumour-directed immune response involving CD8⁺ Tc cells, Th1 cells, and natural killer cells appears to protect against tumour development and progression. In contrast, the immune responses that involve B cells, the activation of chronic humoral immunity and/or a Th2 polarised response, and innate inflammatory cells in the tumour can all promote tumour development and progression (1).

Our findings indicate that early-stage malignancies in dogs may not result in any significant changes in leukocyte subpopulations. However, some of the investigated proteins may become potential biomarkers for metastasis and progression in canine mammary tumours. It can be concluded that lower observed percentages of CD3⁺ T lymphocytes, especially in combination with higher percentages of CD21⁺ B lymphocytes, may indicate an advanced state of disease and the presence of metastases in bitches with mammary tumours. Additionally, our research revealed that in dogs with malignant mammary tumours, the CD4⁺/CD8⁺ ratio decreases gradually with tumour progression and is not suitable as a diagnostic marker for malignancies, but may be a potential biomarker of mammary tumour metastasis.

Our findings may be useful in tumour metastasis detection, but should be considered preliminary because of the small number of blood samples analysed from bitches with mammary cancer. Lymphocyte immunophenotyping in dogs with mammary tumours can provide a deeper understanding of the staging process and, as a result, it can help in improving staging systems in bitches with mammary tumours and possibly in the development of new diagnostic and prognostic markers.