Diagnostic value of combining preoperative inflammatory markers ratios with CA199 for patients with early-stage pancreatic cancer

Pancreatic cancer (PC) is the seventh leading cause of cancer-related deaths in both men and women with nearly equal rates of annual incidence and mortality [1]; it has been projected that by 2030, PC will be the second leading cause of cancer-related deaths, surpassing breast cancer, prostate, and colorectal cancers [2]. Surgical resection remains the primary form of treatment for patients with PC [3]. Currently, the diagnosis of PC is mainly based on clinical signs and symptoms, imaging techniques, serum CA199, and pathological features. However, most patients with PC are already at an advanced stage when they first visit the hospital, losing the opportunity for surgery, with a five-year survival rate of < 5% [4]. Thus, more reliable diagnostic biomarkers are urgently needed to improve early diagnosis of PC.

In recent years, liquid biopsies to isolate circulating tumor DNA (ctDNA) [5], circulating tumor cells (CTCs) [6], circulating exosomal miRNA [7], and exosomal GPC1 [8] for the early detection of PC have re ceived much attention. However, these methods are complex, time-consuming, expensive, and difficult to perform. Tumor-promoting inflammation is the seventh most important feature of cancer cells [9]. Circulating inflammatory markers such as C-reactive protein (CRP) [10], neutrophils [11], lymphocytes, platelets, monocytes [12], and fibrinogen [13] play an essential role in the oncogenesis and development of cancer. Some studies have found that inflammation markers ratios could predict the prognoses of patients with PC. For example, CRP-to-albumin score, the Glasgow Prognostic Score (GPS) each have an independent prognostic value in patients with PC [14]. A high neutrophil-to-lymphocyte ratio (NLR) is associated with an adverse overall survival (OS) in pancreatic cancer [15]. A low fibrinogen-to-albumin ratio (FAR) was positively correlated with a good OS in locally advanced or metastatic PC [16].

Notably, inflammation is evident at the earliest stages of tumor progression and could promote the development of incipient tumors into full-blown cancers [17]. Therefore, we hypothesized that these circulating inflammatory markers change within the early stages of cancer and could act as reliable indicators for early diagnoses of PC. In this study, we assessed inflammation indicator values including FAR, fibrinogen-to-prealbumin ratio (FPR), NLR, platelet-to-lymphocyte ratio (PLR), monocyte-to-lymphocyte ratio (MLR), and prognostic nutritional index (albumin + 5 × lymphocyte count; PNI) in early-stage PC, healthy controls (HC), and other pancreatic tumors (OPT), with the aim of exploring whether inflammation indicators could be used as markers for the diagnosis of early-stage PC.

Cancer-associated inflammation has been reported as the seventh hallmark of cancer [9]. Nearly all human cancers harbor inflammatory reactions, which play an important role in tumor development, progression, and metastasis [18]. Systemic inflammation can play a supporting role in the evolution of PC. For example, chronic pancreatitis is a known risk factor for the development of PC [19]. Obesity, another risk factor for pancreatic cancer, can induce inflammation by promoting the release of IL-6, CCL2, and CCL5, and the infiltration of macrophages and immunosuppressive cells [20]. Smoking is an established risk factor for PC and can induce inflammation and immune activation [21]. In addition, cancer cells can promote systemic inflammation that can, in turn, support tumor growth and lead to a poor prognosis in PC [18]. Inflammatory cells and chemokines shape the inflammatory microenvironment leading to cancer [22]. For example, IL-6, IL-1β, and TNF-α were increased at cancer early stage and associated with disease severity [23]. In Pancreatic ductal adenocarcinoma (PDAC) mouse models, adipocyte-secreted IL-1β could promote obesity-induced pancreatic carcinogenesis and drug resistance through recruitment of tumor-associated neutrophils [24]. High intra-tumoral and serum IL-1β levels in PC patients correlate with poor overall survival and increased chemoresistance [25]. IL-6, a pro-inflammatory cytokine that shows diverse functions of cell multiplication, injury, infection, and inflammation, affects tumor cells to develop PC by controlling vascular endothelial growth factor (VEGF) secretion [26]. IL-8 which derived from macrophages, platelets, and epithelial cells could promote the angiogenesis of PC. Serum levels of IL-6, IL-8, IL-10, and IL-1RA were significantly increased in pancreatic cancer patients and were associated with worse survival rates, poor performance status. A panel of IP-10, IL-6, PDGF plus CA19-9 could discriminate PDAC patients from patients with pancreatic benign disease [27]. TNF-α is associated with acute and chronic inflammation and inflammation related to cancers [28]. In addition, increased expression of tumor-related inflammatory mediators and cytokines, such as TNF-α, IL-1, and IL-6 may stimulate the bone marrow to release neutrophils, resulting in an increase in the circulating neutrophil count and decrease in the circulating lymphocytopenia [29]. Neutrophils could promote growth and metastasis of tumors through secreting a variety of cytokines, including matrix metalloproteinase-9, chemokines and vascular endothelial growth factor (VEGF). It was reported that neutrophils could promote adhesion between circulating tumor cells and distant target organs through acting as an adhesive adapter, finally increasing the chance of distant metastasis. Moreover, neutrophil could also inhibit the antitumor immune function of natural killer cells and cytotoxic T cells [12]. Presently, it is believed that lymphocytes in the peripheral blood can cause synergistic cytotoxicity and play an anti-cancer role. Several subtypes of tumor infiltrating lymphocyte such as CD8 + T cells and memory T cells were associated with better outcomes of a variety of tumors, while regulatory T cells and Th17 cells were associated with progression and unfavorable prognosis of tumors [30]. Although different subset of T cells was associated with adverse prognosis of tumors, high level of absolute lymphocyte count was demonstrated to be associated with favorable prognosis of gastric cancer patients [31]. A study by Dominic et al. showed that inflammatory monocytes were lower in the bone marrow and higher in the blood of patients with resectable PC, and an increased blood-to-bone marrow monocyte ratio was a novel poor prognostic factor for PC [32]. Platelets are also involved in tumor development [33].

Meanwhile, patient’s nutritional status is associated with metabolic changes and immune status impairment. Circulating albumin and prealbumin are markers for evaluating nutritional status and immune status. Albumin can inhibit tumor progression by stabilizing DNA replication and enhancing the immune response [34]. The inflammatory factors may influence nutritional status through inhibition of appetite, alteration of gastrointestinal function, alteration of the carbohydrate metabolism and insulin resistance. Serum levels of IL-6 and IL-8 were inversely correlated to serum albumin and prealbumin. Serum IL-6 and IL-8 were highly expressed in patients with nutritional risk [35]. Genetic and pharmacological studies have revealed the key role of fibrinogen in determining the degree of local or systemic inflammation [36]. Fibrinogen is an important coagulation factor that can be recognized by a variety of integrin and non-integrin receptors on tumor, stromal, and inflammatory cells. These fibrinogen-mediated receptors are thought to control cell proliferation, apoptosis, cell migration, and the expression of inflammatory mediators [37].

In cancer, cytokines mediate signalling between cancer cells, and the cells of the TME, including PSCs, CAFs, endothelial cells, and a range of immune cells including macrophages, mast cells, neutrophils, and regulatory T-cells [38]. For example, glioblastoma (GBM) cells reduced lymphocyte infiltration by secreting immunosuppressive cytokines such as IL-10, IL-2, and TGF-β, and recruited and induced macrophages to become M2 phenotypes by secreting IL-10, IL-4, IL-6, macrophage–colony stimulating factor (M-CSF), TGF-β, and prostaglandin E2 (PGE2) [39]. Higher serum IL-8 and IL-6 levels were positively correlated with high NLR, modified glasgow prognostic score (mGPS), CRP-albumin ratio (CAR), and PLR [40, 41]. Fibrinogen induced the production of IL-6, IL-8, monocyte chemoattractant protein-1, vascular endothelial growth factor, angiopoietin-1 and type I collagen in pancreatic stellate cells [42]. CAR, NLR, and PNI were positively associated with IL-10, IL-23, and IL-1β [43]. Park et al. found moderate-to-strong correlations within circulating cytokines (TNF-α, IL-1β, IL-6, IL-8, IL-9, IL-10, and VEGF) as well as within systemic inflammatory markers (mGPS, NLR, and PNI) [44]. Higher mGPS was involved in increased plasma levels of IL-4, IL-6, IL-8 [45]. Patients with a low PNI exhibited high levels of TNF-αin advanced pancreatic cancer [46]. To sum up, there was a close relationship between systemic inflammatory markers and plasma cytokines.

Currently, routine measurement of serum inflammatory cytokines is not common in daily clinical practice. Many studies used inflammatory cell in the peripheral blood to reflect the systemic immune conditions of patients. In this study, we included HCs and patients with chronic pancreatitis, pancreatic serous/mucinous cystadenoma, solid pseudo-papilloma, and pancreatic neuroendocrine tumors. The results showed that serum albumin, prealbumin, and lymphocytes were significantly decreased, while fibrinogen, neutrophils, and monocytes were significantly increased in early-stage PC compared with HC and OPT. Our results provide supporting evidence that inflammation is emerging as a hallmark of early—stage cancer. Since neutrophil, monocyte, and lymphocyte counts are influenced by many factors, researchers are more inclined to use the ratio values between the two inflammation markers to explore the relationship between the ratio values and malignant tumor prognosis.

To date, many studies have shown that FAR, FPR, NLR, PLR, MLR, and PNI are predictive of outcomes in various types of cancer. For example, Michael et al. [47] found that an increased lymphocyte-to-monocyte ratio (LMR) was an independent prognostic factor for better cancer-specific survival in patients with PC (HR 0.70; P < 0.001). Qi et al. [36] showed that NLR, PLR, and LMR were independent predictors of survival in patients with advanced PC. Yi et al. [46] showed that a low PNI was associated with a systemic inflammatory response and was an independent poor prognostic factor for advanced PC. Fang et al. [16] reported that a high FAR was associated with poor OS in patients with locally advanced or metastatic PC. Xie et al. [48] found that high FPR was an independent poor prognostic factor for patients with stage I-III colorectal cancer (CRC). In addition, inflammatory indicators have important implications in cancer diagnosis. The combination of NLR, PLR, and CEA had a high diagnostic efficacy (AUC = 0.831, 95% CI = 0.807–0.852) for early-stage CRC. Zheng et al. [39] found that NLR + LMR and the derived neutrophil-to-lymphocyte ratio (dNLR) + LMR had good diagnostic performance in patients with glioma (AUC = 0.777 and 0.778, respectively). Wu et al. [49] showed that a combination of PLR and CEA had a better AUC of 0.780 than CEA alone for diagnosing gastric cancer. Lu et al. [50] found that the combination of CA199 and AFR distinguished PC from HC with an ROC of 0.932. Liu et al. [51] showed that combined circulating dNLR and Alb was an effective diagnostic biomarker for early stage PC (AUC = 0.931), and that dNLR could distinguish early-stage PC from HC (AUC 0.895) and from additional cancers (AUC 0.794). Similar to the above results, this study found that FAR, FPR, NLR, PLR, MLR, and FLR were higher in early-stage PC than in HC and OPT, whereas PNI was lower in patients with early-stage PC. These results indicate that inflammatory indicators could act as early diagnostic markers for PC. Moreover, ROC analysis indicated that the FAR, FPR, PLR, MLR, and PNI were promising diagnostic indicators. Among these inflammation markers, a combination of FAR, FPR, FLR, and CA199 could be used to differentiate early-stage PC from HC and OPT with a better AUC (0.964 and 0.924 in training sets). The results obtained in the training set were confirmed for two independent testing sets.

The inflammation indicators were similar over differences in sex, age, tumor size, differentiation, lymph nodes, and TNM stage, but varied greatly for different tumor locations. PHC always obstructs bile ducts, which leads to the levels of albumin (38.2 g/L vs 39.6 g/L, P = 0.007) and prealbumin (207.7 g/L vs 240.1 g/L, P = 0.0003) that are lower than PBTC. We further explored the discriminating ability of inflammation indicators for different tumor locations in early-stage PC. The combination of CA199, FAR, FPR, and FLR could better distinguish PHC from OPHT (AUC = 0.915) than PBTC from OPBTT (AUC = 0.894). Hence, for patients with pancreatic head tumors at the first medical visit, a combination of FAR, FPR, FLR, and CA199 would significantly guide the initial clinical diagnosis and aid in a more accurate final diagnosis.

Our study had some limitations. First, it was a retrospective analysis of data from a clinical trial and lacked prospective data. Second, although all patients were from two single-centers, we enrolled only a small number of patients from one center. Third, the participants in our study had no measurements of serum inflammatory cytokines such as IL-2, IL-6 and so on, we have no way to compared correlations between cytokine levels and inflammation markers ratios. However, despite several limitations, this study confirmed that FAR, FPR, FLR, and CA199 have a potential as diagnostic markers for early-stage PC. These results need to be confirmed in a multicenter, large-scale, prospective study.

This study established that circulating inflammation markers ratios, especially FAR, FPR, and FLR, could be used as cost-effective diagnostic biomarkers for early-stage PC that improve the diagnostic accuracy over CA199. The combination of FAR, FPR, FLR, and CA199 was a potentially effective biomarker for distinguishing early -stage PC patients from HC and in differentiating early -stage PC patients from patients with OPT. The combination of FAR, FPR, FLR, and CA199 may be useful as a differential diagnostic marker for patients with pancreatic head cancer.