Interleukin-6 Differential Expression in Cancer Patients of Different Clinical Stages; a Possible Biomarker of Cancer Progression

Purpose The study investigated interleukin-6 expression pattern across all stages of cancer. The research questions raised in the study were: Is there differential expression of Interleukin-6 across all cancer stages? and what relationship exists between serum interleukin-6 level and cancer stage? Methods The prospective case-control study comprised sixty two (62) purposively selected cancer participants across all stages and age range 18 years to 72years as well equal number of healthy volunteers from two medical centres in Nigeria. Three milliliters (3mls) of blood samples was collected intravenously from the participants and centrifuged after 30 minutes of collection at 3000rpm for 10 minutes to obtain serum. The serum level of Interleukin-6 was determined spectrophotometrically by Enzyme linked immunosorbent assay (ELISA). Data obtained were expressed as mean and standard error of the mean. One way Analysis of variance and t-test were employed to test for signicance difference between the groups and the signicant level was considered at P< 0.05. Findings from the study revealed signicant (P< 0.05) higher mean serum interleukin-6 levels in stage IV cancer participants as compared to other disease stages. In the same way, signicant higher mean Interleukin-6 level of stage III cancer participants as compared to that of stage I cancer participants was observed. Furthermore, the study revealed a signicant correlation (P< 0.01) between serum Interleukin 6 concentration and cancer stage.


Introduction
Cancer, a Public health problem a icting all categories of person is among the global issues, with an estimated 10 million incidences and 6 million annual mortality rates [1]. It is a group of diseases that are characterized by uncontrolled cellular growth, local tissue invasion and distant metastasis caused by aberrations in cellular regulatory mechanisms [2].
Staging of cancer is an important process in cancer evaluation and clinical treatment of solid tumours in which the extent of tumour growth and its spread in the body system are revealed. TNM system is one of the most widely adopted methods of cancer staging which employs histopathological evaluations to assign stages between I and IV on the basis of primary tumour size (T), degree of lymph node involvement (N) and the extent of metastasis (M) [4,25].
Recently, there has been an increasing interest in cancer-related in ammation as a major determinant of disease progression and survival in the eld of oncology research [5,6]. Numerous studies provide evidence that chronic in ammation increases cancer risk, promote tumour progression and support metastasis [7,8].
Interleukin-6 has been subject to increased attention due to its possible usefulness as a biomarker of in ammation [9]. The strong association between in ammation mediated interleukin-6 and cancer development and progression has been well documented [10,11].
Owing to the fact that in ammatory signaling plays key roles in cancer development, progression and therapy response [9], determination and comparison of serum interleukin-6 expression pattern across cancer patients of different disease stage might reveal a better and more reliable predictive prognostic marker as well as identifying target oncogenic mediator that will be of therapeutic importance for the prevention of cancer progression and improved survival outcomes of cancer patients. Therefore, the study investigated interleukin-6 expression pattern in cancer patients of different disease stage from selected Nigeria tertiary health care.

Experimental Design and Participants
The prospective case-control study comprised sixty two (62) purposively selected cancer participants across all stages and age range 18 years to 72years as well equal number of healthy volunteers from two medical centres in Nigerian, namely: University College Hospital (UCH), and Federal Medical Centre (FMC), Idi-aba, Abeokuta.
Participants were included in the study on the basis of absence of neurological disorders and infection, no history of opoid use and they were not on immunosuppressive drugs and anti-in ammatory drugs 24 hrs prior to the study. However, Patients with incidence of pre-existing acute or chronic in ammatory diseases or infectious diseases as well as apparent multiple organ dysfunction were excluded from the study.

Ethical Approval
The approval of the study was obtained from ethics review committee of the selected medical centres with the following ethics committee assigned numbers; UI/EC/19/0161 and FMCA/470/HREC/01/2019/010 respectively. The procedure followed for the conduction of the study were in accordance with the ethical standards of the selected medical institution and with the revised Helsinki Declaration of 1975.
Also, every participant gave their consent prior to the conduction of the study by appending their signatures on the informed consent form.

Participant study
Socio-demographics of the consented participants were collected from the patients using a wellstructured questionnaire.

Clinical Features of the Participants
Participants' clinical information as regard to their cancer stage was retrieved from the hospital. The cancer staging of the participants was based on "TNM" system.

Sample collection
Three milliliters (3mls) of blood samples was collected intravenously from the cancer subjects into a set of well labeled plain sample bottles. The collected blood samples were left standing for 30 minutes after which it was centrifuged at 3000rpm for 10 minutes for serum separation. The collected serum was kept in a Biofreezer set at -20 degree celcius until sample analysis.

Enzyme Linked Immunosorbent Assay (ELISA)
The expression levels of Interleukin 6 cytokines in the separated sera samples were determined spectrophotometrically by Enzyme linked immunosorbent assay using interleukin-6 speci ed ELISA kit as described by Mittal et al [12].

Statistical Analysis
All experiments were set up in triplicates. Data collected were processed using SPSS statistical software version 22.0 and the processed data were expressed as mean ± standard deviation. Analysis of variance and paired t-test were employed to test for signi cance difference among the variables with the level of signi cance considered at P< 0.05 Fig 1.0 shows the percentage age distribution of the participants selected for the study. It was evident from g 1.0 that majority of the cancer patients that participated in the study were of the age groups 40-49 years and 50-59 years with percentage distribution of 32.3% and 30.6% respectively. While minority of the study group comprised of cancer patients of the age group 10-19 years with percentage distribution of 1.6%.    The serum mean Interleukin-6 concentration of the participants with different stages of cancer is presented in Table 3 above. From the table, the mean interleukin-6 concentration increases with advancing stage of disease. However, stage IV participants (with metastatic disease) have signi cant (P< 0.05) higher concentration of Interleukin-6 as compared to other stages. In the same way, signi cant increase in the mean Interleukin-6 concentration of participants with stage III of the disease as compared to that of stage I cancer participants was evident from Table 3.

Discussion
Of recent, there has been an increasing interest in cancer-related in ammation as a major determinant of disease progression and survival in the eld of oncology research. IL6 has been implicated as a member of the major in ammatory interleukins family that has played a signi cant role in various immune, physiological and pathophysiological processes such as cancer progression [13][14][15][16]. Determination of the expression pattern of Interleukin-6, an important in ammatory marker, might reveal a reliable predictive prognostic marker as well as identifying target oncogenic mediator that will be of therapeutic importance for the prevention of cancer progression and improves survival outcomes of cancer patients. Therefore, the present study investigated interleukin-6 expression pattern in cancer patients with different disease stages from selected Nigeria tertiary health care.
Findings from the participants' socio-demographics analysis of this present study revealed Breast cancer and cervical cancer as most common cancer of all the cancer types among the studied participants. This ndings corroborates the ndings of Jedy-Agba et al. in which Breast and cervical cancer were reported as the most common cancers in women from the cancer study conducted in two regions in Nigeria (Table  1) [17]. Also, this nding is in line with the epidemiological and incidence report of common cancers in Nigeria by Abdulkareem in which Breast and Cervical cancer were revealed as rst two most common cancers in Nigeria [18].
Considering the age distribution of the included participants, age group 40-49 years was revealed from the ndings of the study as the most vulnerable age group to cancer disease (Figure 1). The ndings obtained is in line with the work of Akinde et al. [19] on cancer mortality pattern in Lagos University Teaching Hospital, in which age group 41-50 were reported as the most cancer prevalent age groups with percentage distribution of 21.0% respectively [19]. In light of the above, advanced age is an important risk factor of cancer development. Descriptive survey on the cancer stage of the participants in this present study revealed that highest proportions of the participants were at advanced stage (III) of the disease while only small proportion of the studied population were at early stage of cancer. Possible reasons for this observation might be due to late diagnosis and late presentation in the hospitals for cancer treatment.
For the serum Interleukin 6 levels, ndings from this present study revealed that interleukin-6 levels in the serum of the participants diagnosed with advanced cancer stage (III and IV) was signi cantly elevated as compared to that of early stage of the disease. This agrees with earlier reports from a number of studies conducted previously which showed that Patients, who have Cancer have increased levels of IL-6 [20][21][22][23].
Reports from previous studies suggest that an increased production of IL-6 is associated with poor prognosis and survival outcomes in many types of cancers including Breast Cancer [24][25]. Furthermore, our study showed that IL-6 levels were highest in Cancer patients at stage 4, when compared to the other stages in all the Cancer types. Similarly, Chung and Chang reported that an existing association occurred between colorectal tumor size and IL-6 serum levels which peaked at Stage IV in the Patients having colorectal cancer [26].
Conclusively, serum interleukin-6 differential expression in cancer patients at advanced clinical stage as compared to that of early disease stage, alongside with the signi cant association relationship between its expression level and the different clinical stages of cancer as evident from the concluded study. Hence, serum interleukin-6 is highlighted as a promising biomarker of tumour progression and prognosis as well as an important therapeutic target to prevent tumour progression and metastasis for improved survival outcomes of cancer patients. Figure 1 Percentage Age Distribution of the participants. Fig 1 shows the percentage age distribution of the participants selected for the study. It was evident from g 1 that majority of the cancer patients that participated in the study were of the age groups 40-49 years and 50-59 years with percentage distribution of 32.3% and 30.6% respectively. While minority of the study group comprised of cancer patients of the age group 10-19 years with percentage distribution of 1.6%.

Figure 2
Percentage distribution of the participants by cancer stage. Fig 2 revealed the percentage of the cancer patients in different stages of the disease. The gure showed that higher population of the participants involved in the study were in stage 2 and stage 3 of the disease as it was re ected in their percentage distribution of 35.7 % and 38.1% respectively. However, cancer patients at stage 1 of the disease had the lowest percentage (7.1%) of all the recruited participants.