Background: Breast cancer remains an important medical challenge, despite sustained global efforts at its prevention and control. Various immunological factors are expressed in the serum during breast tumourigenesis, and can be of value in the surveillance of the disease. These serum bio-markers include pro-inflammatory cytokines since breast cancer is associated with chronic inflammation. In our locality with different racial/ethnic variations from Caucasian as well as environmental factors, there is scanty information on the value of these serum factors in screening and surveillance of breast cancer—hence the need for this study. Methodology: A total of 68 females (mean age = 48.7 ± 8.7 yrs) with clinically and pathologically confirmed breast cancer were recruited by self selection; representing breast cancer patients group. Due to small sample size they were further grouped into advanced stage breast cancer cases (N = 40) and early stage breast cancer cases (N = 28). Controls consisted of two groups: A—Patient control group (N = 21) comprised females with benign breast tumour (15 cases with fibroadenoma and 6 cases with fibrocystic disease) and group B—apparently healthy age/sex matched control group (N = 21). Pre-treatment samples were collected after which all patients underwent standard treatment modalities (neoadjuvant or adjuvant chemotherapy, radiotherapy, chemoradiation, and/or surgery; depending on the stage of presentation and thereafter post treatment samples were collected after 3 and 6 months respectively. Serum from the patients and controls were assayed immunoenzymatically for TNF-α and IL-1. Results: The results showed that at 6 months post-treatment stage, the mean values of IL-1 differed significantly (P > 0.05) when advanced stage breast cancer were compared with early stage and apparently healthy control groups respectively. Likewise at 6 months post-treatment stage, the TNF-α mean values differed significantly (P > 0.05) between advanced stage breast cancer and apparently healthy control. No significant differences in mean values were recorded across disease and treatment groups in both IL-1 and TNF-α at pre-treatment and 3 months post-treatment stages. Majority of the breast cancer patients studied were married (91%) and had children, of low income, never smoke cigarette, diagnosed at age above 46 years and presented at advanced stages of the disease. Results also showed that 78% of the cases did not have any history of cancer in their families. Also, 63% of the cases had body mass index values suggestive of obesity (>30 kg/m2). Conclusion: Results suggest that the use of serum levels of TNF-α and IL-1 in the diagnosis of breast cancer in our racial/ethnic environment is of limited clinical value. However it could be useful in disease surveillance in metastasis and relapse. Based on our findings, it could also be concluded that cigarette smoking and social sophistication are not among the risk factors to cancer in this part of the world, contrary to the situation in the advanced parts of the world.
The incidence of breast cancer is increasing worldwide [
African countries present with low prevalence, more aggressive, increased mortality, earlier age at presentation (35 - 45 years) and different pattern of gene expression [
The tumour microenvironment is an important aspect of cancer biology that contributes to tumour initiation, tumour progression and responses to therapy. The composition and characteristics of the tumour microenvironment vary widely and are important in determining the anti-tumour immune response [
Tumour necrosis factor (TNF-α) mediates a broad range of biological activities and encompasses beneficial effects for the host in inflammation and in protective immune responses against a variety of infectious pathogens [
Interleukin 1 (IL-1); another inflammatory cytokine produced by activated macrophages with effects similar to that of TNF-α and also helps to activate T cells [
The outcome of breast cancer is usually determined by multiple factors. Serum tumor necrosis factor alpha (TNF-α) concentration has been found to be increased in the circulation of patients with malignancy. Preoperative evaluation of serum TNF-α concentration has been found to be a valuable parameter for reflecting the severity of staging for invasive breast cancer [
Race/ethnicity has been associated with expression of proteins and especially inflammatory cytokines in various diseases [
This is a prospective longitudinal study conducted between February 2015 and June; 2017 on female patients referred to the Oncology Units of Departments of Surgery of University of Nigeria Teaching Hospital, Enugu and Federal Teaching Hospital, Abakaliki (both in Eastern-Nigeria). Diagnosis and staging were clinically and pathologically confirmed.
Sixty eight 68 female breast cancer patients with age range from 30 to 70 years were eligible and recruited for this study; representing breast cancer patients group. Due to small sample size the patients were further divided into early stage breast cancer (stages 1 and 2; N = 28) and advanced stage breast cancer (stages 3 and 4; N = 40) based on Tumour, Node, Metastasis (TNT) classification. Controls consisted of two groups: A―Patient control group: Twenty one (21) females with benign breast tumour (15 cases with fibroadenoma and 6 cases with fibrocystic disease) were involved in this study as benign breast tumour (BBT) patient control group B―apparently age/sex matched healthy control (AHMC) group: A total of 21 healthy females’ volunteers who have no history or clinical evidence of any breast lesions drawn from the Hospital and University Communities were selected as a healthy control group.
Sampling was by self-selection following the approval of the study protocol by the respective Hospitals Ethical Committee, informed written consent obtained from the individuals and exclusion criteria applied. Breast tumour patients who received any therapy prior to diagnosis (surgery/radiotherapy/chemotherapy), previous history of malignancy and history of any other medical illness, which would otherwise limit the survival of the patient in the absence of malignancy, were excluded. All patients underwent standard treatment modalities (neoadjuvant or adjuvant chemotherapy, radiotherapy, chemoradiation, and/or surgery; depending on the stage of presentation. In breast cancer (BC) patients and benign breast tumour (BBT) patients control groups, blood samples were collected before any form of treatment and two more samples at 3 and 6 months interval. In apparently healthy sex/age-matched control, one blood sample was taken from each participant. The samples were allowed to clot, centrifuged at 5000 rpm for 5 minutes, serum separated and stored at −20˚C until analyzed.
Abcam’s TNF-α in vitro Simple StepTM ELISA method were adopted for TNF-α (kit sourced from abcamR, UK, Cat #ab181421) and IL-1a (kit sourced from abcamR, UK, Cat #ab178008) estimations. The simple Step ELISA employs a labeled capture and detector antibody which immunocaptures the sample analyte in solution. This entire complex (capture antibody /detector antibody) is in turn immobilized in the well by immunoaffinity via the anti-tag antibody. Sample or standard are added to wells, followed by the antibody mix. After incubation, the wells are washed to remove unbound material; the TMB substrate is then added. The reaction is stopped by addition of stop Solution which stops the colour development and completes any colour change from blue to yellow. Signal is generated proportionally to the amount of bound analyte and the intensity is measured at 450 nm.
A standard curve was constructed for each method using the respective standard and use for the determination of unknown respective serum sample concentrations.
Data were analyzed using statistical package for Social Sciences (SPSS) software. Statistical significance was set at p < 0.05. Dunn’s multiple comparison tests and Kruskall-Wallis analysis of variance were applied for measuring the differences between disease and treatment groups. GraphPad prism version 6.0 (by GraphPad, USA) was used for the graphs.
Majority of the breast cancer patients studied were married (91%) and had children, of low income, never smoke cigarette, diagnosed at age above 46 years and presented at advanced stages of the disease. Results also showed that 78% of the cases did not have any history of cancer in their families. Also, 63% of the cases had body mass index values suggestive of obesity (>30 kg/m2).
The mean values and statistical comparison of TNF-α and IL-1 in disease groups (breast cancer cases and benign breast tumour) were presented in
Variable | Number (n) | Minimum | Median | Maximum |
---|---|---|---|---|
Age at diagnosis (years) | 68 | 31 | 51 | 69 |
Age at menarche (years) | 65 | 10 | 13 | 16 |
Age of menopause (years) | 42 | 41 | 49 | 55 |
Body mass index (kg/m2) | 68 | 19.2 | 30.8 | 43.5 |
were recorded across disease and treatment groups in both IL-1 and TNF-α at pre-treatment and 3 months post-treatment stages.
No statistical significant difference in TNF-α and IL-1was observed when the breast cancer groups were condensed. Similar results were recorded instead.
Despite the improvement in health facilities; majority of the patients in this study were of low income level and presented at advanced stages of the disease when little or no benefit can be derived from any form of therapy. Out of the 68 breast cancer cases studied, 28 patients presented at early stage (stages 1 and 2), while 40 patients presented at advanced stage of the disease (stages 3 and 4). This could be attributed to poverty, lack of free screening centers and ignorance; giving room for incorporeal interpretation for such health problems. This is indeed worrisome and a major challenge. There is the need therefore for development of measures for early detection of the disease through mass enlightenment and free breast cancer screening at all levels (targeting women organizations and schools) and encouraging regular self-breast examination. The silent nature of breast cancer at onset makes it difficult to be detected early, demanding improved, sensitive and affordable screening procedures. Unfortunately early detection through mammography, routine and self-breast examinations which are so effective in educated communities are seldom applicable in poorly educated ones in whom the carcinomas commonly present late [
Biological markers are widely recognized as important tools in the evaluation and management of patients with cancer. An especially wide array of body fluid markers have been investigated for clinical utility in diagnosing, staging or managing patients with cancer [
Most cancers arise with the association of chronic inflammation and contain inflammatory infiltrates [
The cytokines produced by these cells have been posited as key factors in
Treatment group | Disease stages | Mean (Pg/ml) | Std. Dev | N | Mean diff ± S.E | p-value |
---|---|---|---|---|---|---|
Pretreatment | ESBC | 102.24 | 34.09 | 28 | 9.96 ± 7.29 | 0.175 |
ASBC | 92.29 | 24.03 | 40 | |||
ESBC | 102.24 | 34.09 | 28 | 16.76 ± 9.27 | 0.074 | |
AHMC | 85.49 | 22.32 | 21 | |||
ESBC | 102.24 | 34.09 | 28 | 5.06 ± 8.32 | 0.545 | |
BBT | 97.19 | 36.12 | 21 | |||
ASBC | 92.29 | 24.03 | 40 | 6.80 ± 8.75 | 0.439 | |
AHMC | 85.49 | 22.32 | 21 | |||
ASBC | 92.29 | 24.03 | 40 | 4.90 ± 7.74 | 0.528 | |
BBT | 97.19 | 36.12 | 21 | |||
BBT | 97.19 | 36.12 | 21 | 11.70 ± 9.63 | 0.227 | |
AHMC | 85.49 | 22.32 | 21 | |||
3 months post-treatment | ESBC | 95.64 | 38.89 | 28 | 96.24 ± 56.14 | 0.090 |
ASBC | 191.88 | 366.68 | 38 | |||
ESBC | 95.64 | 38.89 | 28 | 10.15 ± 71.40 | 0.887 | |
AHMC | 85.49 | 22.32 | 21 | |||
ESBC | 95.64 | 38.89 | 28 | 17.19 ± 64.91 | 0.792 | |
BBT | 78.45 | 11.13 | 20 | |||
ASBC | 191.88 | 366.68 | 38 | 106.39 ± 67.39 | 0.118 | |
AHMC | 85.49 | 22.32 | 21 | |||
ASBC | 191.88 | 366.68 | 38 | 113.43 ± 60.47 | 0.064 | |
BBT | 78.45 | 11.13 | 20 | |||
BBT | 78.45 | 11.13 | 20 | 7.04 ± 74.86 | 0.925 | |
AHMC | 85.49 | 22.32 | 21 | |||
6 months post-treatment | ESBC | 93.78 | 22.12 | 26 | 17.03 ± 10.14 | 0.097 |
ASBC | 102.70 | 46.72 | 31 | |||
ESBC | 93.78 | 22.12 | 26 | 8.30 ± 12.52 | 0.051 | |
AHMC | 85.49 | 22.32 | 21 | |||
ESBC | 93.78 | 22.12 | 26 | |||
BBT | - | - | - | |||
ASBC | 102.70 | 46.72 | 31 | 25.33 ± 11.83 | 0.036 | |
AHMC | 85.49 | 22.32 | 21 | |||
ASBC | 102.70 | 46.72 | 31 | |||
BBT | - | - | - | |||
BBT | - | - | - | |||
AHMC | 85.49 | 22.32 | 21 |
Key: ESBC―Early stage breast cancer. ASBC―Advanced stage breast cancer. AHMC―Apparently healthy control. BBT―Benign breast tumour.
Treatment groups | Disease stages | Mean (Pg/ml) | Std. Dev | N | Mean diff ± S.E | p-value |
---|---|---|---|---|---|---|
Pretreatment | ESBC | 56.31 | 29.57 | 28 | 17.17 ± 14.22 | 0.230 |
ASBC | 73.40 | 76.36 | 40 | |||
ESBC | 56.31 | 29.57 | 28 | 10.40 ± 18.08 | 0.566 | |
AHMC | 45.91 | 25.74 | 21 | |||
ESBC | 56.31 | 29.57 | 28 | 24.13 ± 16.24 | 0.140 | |
BBT | 80.44 | 61.03 | 21 | |||
ASBC | 73.40 | 76.36 | 40 | 27.57 ± 17.07 | 0.109 | |
AHMC | 45.91 | 25.74 | 21 | |||
ASBC | 73.40 | 76.36 | 40 | 6.96 ± 15.10 | 0.646 | |
BBT | 80.44 | 61.03 | 21 | |||
BBT | 80.44 | 61.03 | 21 | 34.54 ± 18.78 | 0.069 | |
AHMC | 45.91 | 25.74 | 21 | |||
3 months Post-treatment | ESBC | 77.48 | 61.32 | 28 | 1.94 ± 18.82 | 0.918 |
ASBC | 75.54 | 110.88 | 38 | |||
ESBC | 77.48 | 61.32 | 28 | 31.57 ± 23.93 | 0.190 | |
AHMC | 45.91 | 25.74 | 21 | |||
ESBC | 77.48 | 61.32 | 28 | 33.64 ± 21.76 | 0.125 | |
BBT | 43.83 | 13.60 | 20 | |||
ASBC | 75.54 | 110.88 | 38 | 29.63 ± 22.59 | 0.193 | |
AHMC | 45.91 | 25.74 | 21 | |||
ASBC | 75.54 | 110.88 | 38 | 31.70 ± 20.27 | 0.121 | |
BBT | 43.83 | 13.60 | 20 | |||
BBT | 43.83 | 13.60 | 20 | 2.07 ± 25.09 | 0.934 | |
AHMC | 45.91 | 25.74 | 21 | |||
6 months Post-treatment | ESBC | 59.00 | 39.77 | 26 | 33.37 ± 14.37 | 0.023 |
ASBC | 95.58 | 72.77 | 31 | |||
ESBC | 59.00 | 39.77 | 26 | 13.09 ± 17.73 | 0.463 | |
AHMC | 45.91 | 25.74 | 21 | |||
ESBC | 59.00 | 39.77 | 26 | |||
BBT | - | - | - | |||
ASBC | 95.58 | 72.77 | 31 | 46.47 ± 16.77 | 0.007 | |
AHMC | 45.91 | 25.74 | 21 | |||
ASBC | 95.58 | 72.77 | 31 | |||
BBT | - | - | - | |||
BBT | - | - | - | |||
AHMC | 45.91 | 25.74 | 21 |
Key: ESBC―Early stage breast cancer. ASBC―Advanced stage breast cancer. AHMC―Apparently healthy control. BBT―Benign breast tumour.
modulating immune response either against or in favor of tumourigenesis in the microenvironment. Several pro-inflammatory gene products have been identified that mediate a critical role in suppression of apoptosis, proliferation, angiogenesis, invasion, and metastasis [
Studies in racial/ethnic variations of TNF-α in different disease states have been documented with varied opinions [
Immunohistochemistry reports [
The work of Saganuma [
In this study, there were no significant variations in mean TNF-α values across disease and treatment groups in the pre-treatment and 3 months pos-treatment stages. However at 6 months post-treatment stage, the advanced stage breast cancer mean values deferred significantly with that of apparently healthy control. Higher persistence of TNF-α had been reported previously in metastatic breast cancer [
Interleukin 1 (IL-1); another pro-inflammatory cytokine strongly expressed by monocytes, tissue macrophages, dendritic cells, B lymphocytes and NK cells refers to two proteins encoded by two different genes (IL-1a and IL-1b), both of which share the same cell surface receptors. The IL-1 signaling via its receptors generates local and systemic responses to injury and infection, thereby inducing fever, pain, sensitivity, vasodilatation, hypotension and slow wave sleep; essential processes towards the re-establishment of tissue homeostasis. Prolonged and inappropriate IL-1 induction is shown to be associated with sepsis, rheumatoid arthritis, inflammatory bowel disease, acute myelogenous leukemia, insulin-dependent diabetes mellitus and atherosclerosis [
The role of interleukin-1 (IL-1) as determining factor in the immune and inflammatory responses to tumors cells [
In the present study the serum level of IL-1 was measured in malignant and benign breast diseases and at intervals after treatment to assess the clinical utility as a tumour marker. No significant difference was observed across the disease and treatment groups in pre-treatment and 3 months post-treatment. However at 6 months pre-treatment stage, the advanced breast cancer IL-1values differed significantly when compared with early stage and apparently healthy control. Higher persisted of IL-1 had been reported in metastatic breast cancer [
The study of Soria [
The discrepancies between this study and the previous studies may lie in the fact that all our patients were of Negroid black race with different MBL polymorphism, in contrast to racial/ethnic different Caucasians. Though there is no known study on the MBL polymorphism in our environment; the polymorphism in MBL and MASP-2 gene is suggested to affect the serum concentration of MBL and MASP-2 [
Breast cancer remains intractable and common among women, possible potentials of these biomarkers in early diagnosis, prognosis and monitoring of treatment is of immense public health importance. Since there has not been any known work in molecular profile of tumour markers in women from the target population; this study has provided a ground breaking attempt from where other studies can be conducted.
The prime limitation encountered was that of follow up for the repeat sampling after various forms of treatment because some patients resorted to unorthodox and spiritual healingand some traveled overseas or died. Some patients therefore, were lost to follow-up and that affected the comparison after six months pos-treatment.
Felix, C.E., Ehigiator, I.F. and Chinedum, C.F. (2018) Pro-Inflammatory Cytokines (TNF-α and IL-1) in Nigerian Women with Breast Cancer. Open Journal of Immunology, 8, 13-28. https://doi.org/10.4236/oji.2018.82002