Prognostic value of cytokines in breast cancer: Correlation with positive hormonal status and obesity

Breast cancer is one of the most common cancers in female leading to cancer-related death^[1]. The course of the disease is affected my many variables including initial TNM staging, hormone receptor status, and Her2 expression^[2].

Interleukin 6 (IL6) is a cytokine producing low-grade inflammation inducing cancer. It is also one of the cytokines released in obese patients^[3].

IL6 was associated with the TNM staging^[4] especially axillary lymph node affection^{[5, 6]} and also with the extent and survival in the metastatic setting^[7]. It was also related to prognosis in triple-negative breast cancer (TNBC)^[8].

Interferon gamma (IFγ) is a cytokine affecting outcome in breast cancer^[9]. It has two contradictory effects in cancer cells. It has shown direct anti-tumor activity. However, this cytokine has been also involved in the activation of programmed cell death (PDL1). This leads to protection of the cancer cells from the immune attack which ultimately leads to tumor growth. Anti-PDL1 may be considered in such patients^[10]. High level of IFγ may affect the response in estrogen-positive breast cancer^{[11, 12]}.

Obesity is rising in many countries^[13]. Adipose tissue is associated with pro-inflammatory cytokines, such as IL6, which may affect the prognosis and response to hormonal treatment^{[14, 15, 16]}. Whereas IFγ is not well studied in breast cancer patients, correlation between the two cytokines is of benefit as there are some data on IL6 with only scarce information on IFγ in breast cancer.

All breast cancer patients with estrogen receptor (ER) positive and or progesterone receptor (PR) positive were recruited prior to chemotherapy and were classified according to body mass index into normal (BMI 18.5–24.9), overweight (25–29.9), or obese (BMI ≥ 30). They were also stratified into metastatic and non-metastatic.

The study included only luminal A and luminal B molecular subtypes. Other subtypes including HER2 enriched and basal triple-negative breast cancer (TNBC) were not included.

Luminal A was ER and PR positive with low Ki67 and HER2 negative. Luminal B was ER and PR positive with high Ki67, and HER2 may be negative or positive. HER2 enriched is a high expression of HER2 with negative ER and PR. Basal type is low expression of ER, PR, and HER2^[17,18]. Basal type is known as triple-negative breast cancer.

Correlation between different cytokines with the TNM staging, hormonal status, Her2, and disease progression as well as response to treatment will be evaluated.

Venous blood sample was drawn from 70 hormone receptor-positive breast cancer patients at the time of diagnosis before the initiation of systemic therapy. This included all stages, metastatic and non-metastatic.

Serum levels of IL-6 were determined with an enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, MN, USA; Quantikine human IL-6). The detection limit of the assay was 0.7 pg/ml. Within-assay reproducibility has been tested before.

All data were statistically studied by descriptive analysis. The level of cytokines was correlated with the different clinico-epidemiological factors. This included the initial TNM staging, hormonal status, Her2 status, and BMI.

Data were statistically described in terms of mean ± standard deviation (± SD), median, and range, or frequencies (number of cases) and percentages when appropriate. Correlation between various variables was done using Spearman rank correlation equation. P value less than 0.05 was considered statistically significant. All statistical calculations were done using computer program IBM SPSS (Statistical Package for the Social Science; IBM Corp, Armonk, NY, USA) release 22 for Microsoft Windows.

The clinico-epidemiological characteristics of all 78 breast cancer patients included in the study are shown in Table 1. The median age at diagnosis was 54 years. The majority of the cases were T2 (62.8%), N1 (38.5%), and M0 (89.74%) with stage II being the most common (47.4%). Most females were estrogen receptor (97.9%) and progesterone receptor positive (96.9%) with high Ki67 ≥ 20 (61.5%). Her2 neu positive presented 16.7%. Luminal A and luminal B presented 29.5% and 53.8%, respectively. Patients with triple-negative breast cancer were not included in the study. Concerning the body mass index, obese patients presented by far the majority (82.1%), and overweight and normal weight constituted only 10.2% and 7.7%, respectively.

The correlation of IL6 and IFγ with various prognostic factor is shown in Table 2. The median serum level IL6 was 56.20 ± 28.715, whereas IFγ was 76.37 ± 41.54. The level of IL6 was significantly correlated with tumor size (P = 0.001), nodal involvement (P = >0.0001), and the presence of metastasis (P = 0.008). Thereby the stage was affected (P = >0.0001). High level of IL6 was associated with positive estrogen receptor, Her2 neu enriched, luminal A, and being obese (P = 0.09, 0.07, 0.06, and 0.05, respectively).

Concerning IFγ, a high serum level was only associated with lower nodal burden being significantly higher in N1 than in N3 (118.15 ± 31.07 vs 76.37 ± 44.46, P = 0.01) and early stage (P = 0.02).

The median level of IL6 was 58.39 and was significantly increased with the tumor size (P = 0.001), nodal involvement (P = >0.0001), and the presence of metastasis (P = 0.008).

This was in accordance with other study by Saeed et al. (2019) that found the level of IL6 was significantly increased with TNM staging^[19].

This result was also consistent with Ravishankaran and Karunanithi (2011), where the level of IL6 was monitored in 59 female breast cancer patients prior to surgery. There was a significantly high level of IL6 with lymph node metastasis and TNM staging, and this was correlated with poor overall survival (P = 0.003)^[4].

Our results were congruous with the study of Noman et al. (2017), which was done on 110 female breast cancer patients and 30 volunteers. Long-term follow-up of patients showed an association between the elevated IL6 and the risk of bone metastasis as well as early recurrence^[20]. In a big Chinese study performed on 534 individuals, IL6 was also a poor prognostic marker for disease progression and metastasis^[5].

In the present study, elevated level of IL6 was associated with estrogen receptor positive and HER2/neu enrichment (P = 0.09, P = 0.09). This was in accordance with Huang et al. (2016), where IL6 in HER2-positive breast cancer leads to drug resistance to lapatinib. IL6 has been proved to be a regulatory cytokine with high level leading to tumor progression^[21].

The level of IL6 was increased with high body mass index and was significantly increased in obese breast cancer patients (P = 0.05). This is in agreement with other studies^[14,15,16].

Inflammation of mammary adipose tissue occurs in overweight and obese patients exhibiting early-stage breast cancer. This was evident in the study by Vaysse et al. (2017) on 107 breast cancer patients^{[22, 23]}.

The IFγ is a pro-inflammatory cytokine produced by T helper 1 cell (Th1). It is responsible for cell-mediated immunity. It was not well studied before. To the best of our knowledge, this study included the largest sample in breast cancer.

IFγ level was inversely correlated to the nodal status (P = 0.01) and the staging (P = 0.02). This was in agreement with Zhu et al. (2013) investigating the cytokines as they regulate the immune function and thereby improve the treatment outcome in breast cancer patients. The ratio of IFγ/IL6 was in node negative patients compared to the positive ones (P = 0.012). This was associated with lower stage (P = 0.068) and was reflected by better treatment outcome and improved survival^[9].

New studies treating patients with IFγ along with chemotherapy resulted in longer disease-free survival. There is an ongoing trial with paclitaxel, trastuzumab, and pertuzumab in Her2/neu-positive patients. Data are not yet available^[24].

IFγ is related to the activation of the immune system. This is the hypothesis that was investigated in breast cancer clinical trials where immunotherapy was added to chemotherapy and target therapy. It was also added in other types of cancer such as colon, ovary, and glioblastoma^{[24, 25]}.

IFγ treatment in HER2 breast cancer in mice has shown a synergistic effect when combined with target therapy and immunotherapy, so when combined with anti-HER2 neu and anti-PDL 1, the response to therapy is improved^[26].

IFγ was not associated with any molecular subtype (P = 0.498). Moreover, it was not affected by the body mass index of the patients (P = 0.766). This is in accordance to a recent study showing that ER positive is associated with IFγ if it is signaling with Δ133TP53 RNA in mutant TP53 (mTP53) and ER negative if IFγ is signaling TP63 RNA in wild-type TP53 (wtTP53). IFγ was associated with central obesity in normal female^{[27, 28]}.

The sample size of the patients was small and requires longer follow-up.

IL6 level was correlated to the initial staging of the patients. This includes the tumor size, lymph node status, and metastasis. It was also related to hormonal status, Her2 positive, and obesity. The IFγ level was inversely correlated to the lymph nodal status and initial staging. The levels of IL6 and IFγ were inversely correlated together regarding the nodal status (P = 0.05).