Serum pro-inflammatory cytokines as potential biomarkers for the diagnosis of gastric carcinoma

Abstract

BACKGROUND

Multiple lines of evidence have indicated that pro-inflammatory cytokines play a role in the pathophysiology of gastric carcinoma (GC).

AIM

To identify potential serum cytokine-based biomarkers for GC diagnosis.

METHODS

The study cohort comprised 50 patients diagnosed with GC and 50 healthy control subjects. A panel of 7 pro-inflammatory cytokines, including interleukin (IL)-1β, IL-2, IL-6, IL-8, IL-12, tumor necrosis factor-α, and interferon-γ (IFN-γ) were quantified using multiplex Luminex assays. Comparative analyses were conducted to evaluate cytokine levels between the GC patients and healthy controls. The diagnostic potential of serum pro-inflammatory cytokines in differentiating GC patients from healthy individuals was assessed through receiver operating characteristic (ROC) curve analysis. The correlation between serum cytokine levels and disease severity, as classified by the tumor-node-metastasis staging system, was analyzed using Spearman's rank correlation coefficient.

RESULTS

In comparison to the control group, patients with GC demonstrated significantly elevated serum levels of IL-1β (t = -4.089, P < 0.001), IL-6 (t = -3.983, P < 0.001), IL-8 (t = -5.460, P < 0.001), and IFN-γ (t = -2.856, P = 0.005). ROC curve analysis indicated that the area under the curve values for IL-1β, IL-6, and IL-8 exceeded 0.7, effectively distinguishing GC patients from healthy controls. Additionally, serum levels of IL-1β (r = 0.424, P = 0.012) and IL-6 (r = 0.742, P < 0.001) were positively correlated with the T stage in GC patients. Similarly, serum concentrations of IL-1β (r = 0.356, P = 0.039) and IL-6 (r = 0.441, P = 0.008) exhibited a positive association with the N stage in these patients.

CONCLUSION

These findings suggest that circulating pro-inflammatory cytokines, such as IL-1β, IL-6, and IL-8, may serve as potential biomarkers for the diagnosis of GC.

Key Words: Gastric carcinoma; Pro-inflammatory cytokines; Serum; Biomarker; Diagnosis

Core Tip: This investigation compared the concentrations of seven pro-inflammatory cytokines between gastric carcinoma (GC) patients and healthy controls, aiming to identify serum cytokine-based biomarkers for GC. The study produced four main findings. Firstly, GC patients demonstrated elevated levels of interleukin (IL)-1β, IL-6, IL-8, and interferon-γ compared to controls. Secondly, receiver operating characteristic curve analysis indicated that the area under the curve values for IL-1β, IL-6, and IL-8 exceeded 0.7 in differentiating GC patients from healthy individuals. Thirdly, serum levels of IL-1β and IL-6 showed a positive correlation with the T stage of the disease. Fourthly, the serum concentrations of IL-1β and IL-6 were positively associated with the N stage. These findings suggest that circulating pro-inflammatory cytokines, such as IL-1β, IL-6, and IL-8, may serve as potential biomarkers for the diagnosis of GC.

INTRODUCTION

Gastric carcinoma (GC) ranks as the second most frequently diagnosed cancer in China and constitutes the third leading cause of cancer-related mortality globally[1]. The diagnosis of GC is frequently delayed in numerous patients due to the absence of specific symptoms. Patients presenting with advanced stages of the disease are often ineligible for surgical intervention, resulting in a poor 5-year survival rate of approximately 25%[2,3]. Early detection and appropriate treatment, guided by precise risk stratification, are essential for enhancing the prognosis of GC. Currently, endoscopic mucosal biopsy is regarded as the gold standard for the diagnosis of GC. Due to its high cost and invasive nature, widespread adoption in China is challenging. Additionally, current tumor markers, including carcinoembryonic antigen, carbohydrate antigen (CA) 199 (CA199), and CA724, lack adequate sensitivity and specificity for the diagnosis of GC[4]. Thus, there is an imperative need to identify novel and reliable non-invasive biomarkers for the detection of GC, which represents a critical step toward early intervention and the reduction of mortality rates.

The interplay between inflammation and cancer has been a focal point of research for an extended period[5,6]. The inflammatory tumor microenvironment consists of inflammatory cells, chemokines, cytokines, and signaling pathways, with inflammatory cytokines playing a particularly pivotal role in cancer development, prognosis, and treatment[7,8]. Pro-inflammatory cytokines are associated with advanced cancer stages, resistance to immunotherapy, and unfavorable prognoses, including reduced objective response and disease control rates, as well as decreased progression-free and overall survival[9]. In terms of GC, pro-inflammatory cytokines such as interleukin (IL)-1β, IL-6, and interferon-γ (IFN-γ) have been associated with an increased risk of GC[10]. Moreover, IL-6, a pro-inflammatory cytokine, facilitates the growth and progression of GC[11]. Furthermore, the administration of tocilizumab, an anti-human IL-6 receptor antibody, has been shown to enhance the anti-tumor efficacy of chemotherapy in GC[12]. Additionally, a study conducted in Italy suggests that IL-1β, IL-8, and tumor necrosis factor-α (TNF-α) may serve as diagnostic markers in patients with GC[13]. These findings suggest that peripheral cytokines are implicated in the pathophysiology of GC and may hold significant potential as diagnostic biomarkers for the disease.

In this study, we investigated the serum concentrations of various pro-inflammatory cytokines in patients with GC with two primary objectives: (1) To identify potential serum biomarkers for the diagnosis of GC, and (2) To assess the relationship between serum pro-inflammatory cytokine levels and disease severity in GC patients.

MATERIALS AND METHODS

Subjects

A cohort of 50 patients diagnosed with GC and admitted to the Second People's Hospital of Hefei, China, between November 2023 and October 2024, were recruited for this study. Each patient presented with upper abdominal discomfort and received a GC diagnosis confirmed through pathological examination. Concurrently, a control group comprising healthy individuals was established. These control subjects were volunteers who participated in a complimentary health screening designed to identify any potential organic lesions in the stomach. Clinical data were extracted from the medical records of the participants. The exclusion criteria were: (1) Individuals undergoing radiotherapy or chemotherapy; (2) Individuals diagnosed with other forms of cancer or major organ diseases, including those affecting the heart, liver, kidneys, or lungs; (3) Individuals with severe active infectious diseases; and (4) Individuals with severe hematological disorders, those who have undergone bone marrow transplantation, or those with severe trauma or immune disorders. The control group included 50 healthy volunteers, matched according to age, gender, and body mass index (BMI). In accordance with the guidelines established in the Declaration of Helsinki, all participants in the study provided informed written consent prior to their involvement. Approval for the study was granted by the Ethics Committee of Second People's Hospital of Hefei (Approval No. 2023-keyan-123).

Sample collection and preparation

The blood sample was collected from the participant's vein following a 10-11 hour overnight fasting period, between 7:00 a.m. and 8:00 a.m. Subsequently, following centrifugation at 12000 g for 5 minutes at 4 °C, the resulting supernatant was utilized as serum samples. These serum samples were stored at -80 °C until further analysis. Moreover, the control samples were matched as closely as possible to the GC group with respect to the time period of sample collection.

Measurement of serum cytokines

The measurement method was performed according to a previous study[14]. A panel of 7 cytokines, including IL-1β, IL-2, IL-6, IL-8, IL-12, TNF-α, and IFN-γ, was quantified using multiplex bead immunoassays (LXSAHM-10 and LXSAHM-27, R&D Systems for Antibody Detection, Shanghai Universal Biotech Co., Ltd.) according to the manufacturer's instructions. Standard curves were established using the reference cytokine samples provided in the kit, facilitating the determination of cytokine concentrations in aqueous humor samples. All values were within the calibration curve range. To ensure that the results were not unduly influenced by a single data point and as is the convention, outliers (datapoint > 3 SD above or below the mean) were removed from the dataset.

Sample calculation

The sample size for this study was determined using G*Power software version 3.1.9.6 according to a previous study[15]. For an independent t-test (one-tailed) with a medium effect size of 0.5, a significance level of 0.05, and a power of 0.80, the minimum required sample size was calculated to be 53 for each group, for a total of 106 participants.

Statistical analysis

Data analysis was performed using SPSS software (Version 17.0; SPSS, Inc., Chicago, IL, United States). The normality of data distributions was assessed using the Kolmogorov-Smirnov one-sample test. For variables with normal distributions, comparisons between groups were conducted using the independent samples t-test, whereas the Mann-Whitney U test was employed for variables that did not exhibit normal distribution. Receiver operating characteristic (ROC) curve analysis was conducted to evaluate the area under the curve (AUC) of serum pro-inflammatory cytokines for differentiating GC patients from healthy controls. Correlation analyses were performed using the Spearman correlation test. A two-tailed P-value of less than 0.05 was deemed indicative of statistical significance.

RESULTS

Demographic characteristics of the participants

The demographic characteristics of the study participants are detailed in Table 1. No statistically significant differences were observed in age (t = -1.031, P = 0.305), sex (χ² = 0.000, P = 1.000), or BMI (t = -1.227, P = 0.224) between GC patients and healthy controls.

Table 1 Comparison of demographic data between control group and gastric carcinoma group, mean ± SD.

	Control group	GC group	t/χ2	P value
Age	69.76 ± 10.59	71.84 ± 9.56	-1.031	0.305
Gender (female/male)	15/35	15/35	0.000	1.000
BMI (kg/m²)	22.08 ± 4.01	23.25 ± 3.42	-1.227	0.224

GC: Gastric carcinoma; BMI: Body mass index.

Comparison of serum pro-inflammatory cytokines between patients with GC and healthy controls

Figure 1 illustrates the serum levels of pro-inflammatory cytokines in GC patients and healthy controls. Compared to the controls, GC patients demonstrated significantly elevated levels of IL-1β (t = -4.089, P < 0.001), IL-6 (t = -3.983, P < 0.001), IL-8 (t = -5.460, P < 0.001), and IFN-γ (t = -2.856, P = 0.005). In contrast, no significant differences were observed in the levels of other cytokines, including IL-2 (t = -0.583, P = 0.561), IL-12 (t = 0.223, P = 0.824), or TNF-α (t = -0.829, P = 0.409), between the two groups.

Figure 1 Comparison of serum pro-inflammatory cytokines between control group and gastric carcinoma group. A: Comparison of interleukin (IL)-1β; B: Comparison of IL-2; C: Comparison of IL-6; D: Comparison of IL-8; E: Comparison of IL-12; F: Comparison of tumor necrosis factor-alpha; G: Comparison of interferon-γ. The data are presented as the mean ± SD. ^aP < 0.01 vs the control group, NS: No significance. GC: Gastric carcinoma.

Diagnostic efficacy of serum pro-inflammatory cytokines in distinguishing patients with GC from healthy controls

The diagnostic efficacy of various pro-inflammatory cytokines in distinguishing GC patients from healthy controls was assessed using ROC curve analysis (Figure 2). The analysis revealed AUC values of 0.708 for IL-1β, 0.717 for IL-6, 0.784 for IL-8, and 0.647 for IFN-γ. Notably, the AUCs for IL-1β, IL-6, and IL-8 exceeded 0.7, suggesting these cytokines possess substantial diagnostic value. Specifically, for IL-1β, a cut-off value of 5.665 pg/mL yielded a sensitivity of 70% and a specificity of 64%; for IL-6, a cut-off of 2.850 pg/mL resulted in a sensitivity of 76% and a specificity of 64%; for IL-8, a cut-off of 13.050 pg/mL provided a sensitivity of 74% and a specificity of 72%; and for IFN-γ, a cut-off of 10.950 pg/mL achieved a sensitivity of 52% and a specificity of 72%. A combined panel of IL-1β, IL-6, IL-8, and IFN-γ exhibited high diagnostic accuracy in differentiating GC patients from healthy controls, with an AUC of 0.888.

Figure 2 receiver operating characteristic curves of differential pro-inflammatory cytokines in identification of patients with gastric carcinoma from healthy volunteers. A: Receiver operating characteristic (ROC) curve of interleukin (IL)-1β; B: ROC curve of IL-6; C: ROC curve of IL-8; D: ROC curve of interferon-γ (IFN-γ); E: ROC curve of a combined panel of IL-1β, IL-6, IL-8, and IFN-γ. IL: Interleukin; AUC: Area under the curve; IFN-γ: Interferon-γ.

Association between the serum levels of pro-inflammatory cytokines and the disease severity in patients with GC

The tumor-node-metastasis (TNM) classification system is the globally recognized standard for evaluating the extent of tumor dissemination in GC. Figure 3 illustrates a correlation plot that examines the relationship between serum concentrations of pro-inflammatory cytokines and TNM classification in patients diagnosed with GC. The analysis revealed a positive correlation between serum levels of IL-1β (r = 0.424, P = 0.012) and IL-6 (r = 0.742, P < 0.001) with the T stage. Additionally, serum levels of IL-1β (r = 0.356, P = 0.039) and IL-6 (r = 0.441, P = 0.008) were positively correlated with the N stage.

Figure 3 Correlation between the serum levels of differential pro-inflammatory cytokines and the disease severity in patients with gastric carcinoma. ×: No significance; IL: Interleukin; IFN-γ: Interferon-γ.

DISCUSSION

This investigation compared the concentrations of seven pro-inflammatory cytokines between GC patients and healthy controls, aiming to identify serum cytokine-based biomarkers for GC. The study produced four main findings. Firstly, GC patients demonstrated elevated levels of IL-1β, IL-6, IL-8, and IFN-γ compared to controls. Secondly, ROC curve analysis indicated that the AUC values for IL-1β, IL-6, and IL-8 exceeded 0.7 in differentiating GC patients from healthy individuals. Thirdly, serum levels of IL-1β and IL-6 showed a positive correlation with the T stage of the disease. Fourthly, the serum concentrations of IL-1β and IL-6 were positively associated with the N stage.

Cytokines are recognized for their substantial impact on the initiation and progression of various cancers, including GC. However, the specific mechanisms underlying this influence remain inadequately understood and may be associated with Helicobacter pylori (H. pylori) infection. Notably, approximately 90% of GC cases have been linked to H. pylori infection[16]. In the case of GC, the chronic inflammatory condition of the stomach induced by H. pylori infection, along with the production of inflammatory mediators such as cytokines and chemokines within gastric tissues, is hypothesized to play a critical role in the development and progression of the disease[17]. On one hand, H. pylori interacts with surface epithelial cells, causing direct cellular damage or inducing the production of pro-inflammatory mediators[18]. On the other hand, H. pylori penetrates the underlying mucosa to stimulate an immune response, resulting in the release of various cytokines and oxygen radicals that can transform chronic gastritis into gastroduodenal ulcers and GC[19]. Therefore, this study seeks to identify serum pro-inflammatory cytokine-based biomarkers for GC.

The selection of these seven pro-inflammatory factors including IL-1β, IL-2, IL-6, IL-8, IL-12, TNF-α, and IFN-γ as potential biomarkers for GC diagnosis is predicated on three primary considerations. Firstly, their involvement in the carcinogenesis process is well-documented. A substantial body of research indicates that inflammation is integral to various stages of tumor initiation, progression, invasion, and metastasis. These pro-inflammatory factors are implicated in the interplay between inflammatory responses and tumor dynamics. For instance, IL-1β is known to facilitate cell proliferation and inhibit apoptosis by activating specific signaling pathways, thereby creating an environment conducive to cancer cell growth[20]. Similarly, TNF-α is involved in the regulation of angiogenesis-related factors, promoting tumor angiogenesis and thus supplying the necessary nutrients for tumor growth and metastasis[21]. Secondly, the aberrant expression of these factors in patients with GC is noteworthy. Compared to healthy individuals or patients without GC, the levels of these pro-inflammatory factors in patients with GC exhibit significant alterations. Clinical investigations have demonstrated a significant upregulation in the expression levels of factors such as IL-6 and IL-8 in the serum or tumor tissues of patients with GC[22]. These aberrant expressions are closely associated with the stage of GC, the pathological type, and patient prognosis. Given the critical role these pro-inflammatory factors play in the progression of GC, they may serve not only as diagnostic markers but also as potential therapeutic targets. Monitoring the levels of these factors could facilitate the evaluation of treatment efficacy and the prediction of patient prognosis. For instance, targeted therapeutic agents against TNF-α have shown some efficacy in the treatment of certain cancers[23,24].

IL-1β is a pro-inflammatory cytokine that plays a pivotal role in mediating inflammation and immune responses. Research has shown that infection with Helicobacter pylori activates the inflammasome, resulting in the overexpression and secretion of IL-1β by neutrophils, macrophages, and dendritic cells within the gastric mucosa[25]. In conjunction with H. pylori infection, IL-1β emerges as an indispensable factor in the onset and progression of GC[26,27]. IL-1β is strongly associated with severe gastric inflammation, the development of GC, and poor prognosis[28,29]. The association between IL-1β and GC is facilitated through several mechanisms, including inflammatory immune damage, suppression of gastric acid secretion, aggregation of bone marrow-derived cells, promotion of angiogenesis, and enhancement of gastric cancer cell invasion and metastasis[30]. A recent study reported significantly elevated serum levels of IL-1β in patients with GC compared to healthy individuals[31]. In line with these findings, the present study also observed higher serum levels of IL-1β in GC patients relative to controls. Moreover, serum IL-1β levels were positively correlated with both T stage and N stage. These findings contribute additional evidence linking IL-1β to the pathophysiology of GC.

IL-6 is a soluble mediator with pleiotropic effects on inflammation, immune response, and hematopoiesis. It is rapidly and transiently produced in response to infections and tissue injuries, contributing to host defense by stimulating acute phase responses, hematopoiesis, and immune reactions[32]. Recent research has implicated IL-6 in the initiation and progression of tumors[33]. Multiple lines of evidence identifies IL-6 as a critical factor in GC[12,34]. Previous studies have demonstrated a significant correlation between systemic IL-6 Levels induced by H. pylori infection and the incidence of GC[35]. H. pylori infection facilitates an IL-6-mediated autocrine and paracrine positive feedback loop between macrophages and gastric epithelial cells, which may contribute to GC[36]. Clinical data indicate that elevated serum IL-6 levels are observed in GC patients and are associated with disease progression and poorer prognosis[37]. Experimental findings suggest that IL-6 enhances the proliferation and invasiveness of GC cell lines[38]. Conversely, IL-6 knockout mice exhibit a lower incidence of GC and reduced tumor size[39]. Collectively, these findings indicate that an exaggerated IL-6 response may contribute to the pathogenesis of GC. In the current study, elevated serum levels of IL-6 were observed in patients with GC and were positively correlated with both T stage and N stage.

IL-8, a pro-inflammatory cytokine belonging to the CXC chemokine family, has recently been identified as playing a pivotal role in cancer invasion, angiogenesis, and metastasis, and is regarded as a significant component of the tumor microenvironment[40-42]. Stromal cells are capable of producing IL-8, thereby influencing the invasive or metastatic potential of cancer cells, while cancer cells themselves can secrete IL-8 through autocrine or paracrine mechanisms[43]. IL-8 has been extensively implicated in the carcinogenesis of GC. Studies have reported that IL-8 expression is significantly upregulated in GC tissues compared to paired normal control tissues, with this increased expression being strongly associated with poorer overall prognosis[44,45]. Moreover, IL-8 overexpression enhances the adhesion, migration, invasion, and chemoresistance of GC cells[46]. Furthermore, a meta-analysis has identified high IL-8 expression in either the tissues or serum of GC patients as an independent risk factor influencing the prognosis of GC[47]. Consistent with these findings, the present study also noted elevated serum IL-8 Levels in GC patients, indicating that circulating IL-8 Levels may serve as a potential biomarker for GC diagnosis.

IFN-γ, a multifunctional cytokine, is primarily produced by T helper cells, cytotoxic T cells, natural killer cells, and macrophages during the initial phase of infection. Recent studies have reported an upregulation of IFN-γ in the gastric mucosa following chronic H. pylori infection[48]. Moreover, IFN-γ has been shown to be upregulated in resected GC tissues compared to matched adjacent noncancerous tissues[49]. Experimental evidence suggests that IFN-γ may promote GC cell proliferation and metastasis, partially through the upregulation of integrin β3-mediated nuclear factor kappa B (NF-κB) signaling[50]. Clinical studies have demonstrated that circulating levels of IFN-γ are significantly higher in GC patients compared to healthy controls[51]. Similarly, in the present study, patients with GC demonstrated elevated serum levels of IFN-γ compared to the control group.

To evaluate the potential of these aberrant pro-inflammatory cytokines as diagnostic biomarkers for GC, a ROC analysis was performed. According to established criteria, an AUC value exceeding 0.7 in ROC analysis is deemed clinically significant for screening purposes[52,53]. Three pro-inflammatory cytokines, specifically IL-1β, IL-6, and IL-8, met this criterion. Previous studies have shown that the combined detection of multiple serum proteins as a single panel can improve sensitivity or specificity compared to individual biomarkers[54]. Therefore, we incorporated a combination of IL-1β, IL-6, IL-8, and IFN-γ for the diagnosis of GC. The results demonstrated that the AUC value of this panel increased to 0.888, indicating that this panel shows significant promise as a biomarker for identifying patients with GC.

The TNM classification system, an abbreviation for Tumor, Node, Metastasis, is a globally recognized framework that delineates the extent of cancer dissemination[55]. To investigate the correlation between serum levels of pro-inflammatory cytokines and disease severity in patients with GC, we utilized Spearman's rank correlation coefficient to evaluate the association between four aberrant pro-inflammatory cytokines and the TNM classification in individuals diagnosed with GC. The findings revealed that serum levels of IL-1β and IL-6 demonstrated a positive correlation with the T stage, while serum levels of IL-1β and IL-6 were also positively associated with the N stage. Given the cross-sectional nature of this study, further research is required to clarify the causal relationship between these pro-inflammatory cytokines and the TNM classification in GC patients.

This study is subject to several limitations. Firstly, the relatively small sample size and the data collection from a single center may introduce potential bias into the findings. Secondly, it is not possible to establish causal relationships between disease severity and the observed aberrations in pro-inflammatory cytokines based on the study due to its cross-sectional design. Thirdly, the study did not assess the dynamic changes in pro-inflammatory cytokines across different stages of GC. Fourthly, another limitation of our study is the long period (almost 1 year) over which the samples were collected.

CONCLUSION

In conclusion, this study identifies three pro-inflammatory cytokines, namely IL-1β, IL-6, and IL-8, as potential diagnostic biomarkers for distinguishing patients with GC. To validate their clinical utility as diagnostic indicators for GC, further multicentric studies are warranted. Moreover, a longitudinal research design is necessary to investigate potential causal relationships between aberrant pro-inflammatory cytokine levels and the development of GC.