Application of inflammatory markers in the diagnosis and prognosis of colorectal cancer

Abstract

This invention provides an application of inflammatory biomarkers in the diagnosis and prognosis of colorectal cancer. This invention discovers that systemic inflammatory biomarkers are associated with the progression of colorectal cancer; one or more of NLR, LMR, PLR, SIRI, RDW, SII, and CEA have superior diagnostic value compared to other indicators, exhibiting good diagnostic value and effectively distinguishing colorectal cancer patients from healthy individuals and adenoma patients; among them, the combined use of SIRI, RDW, and CEA, and the combined use of LMR and RDW, are good biomarkers for diagnosing colorectal cancer; furthermore, RDW and CEA can serve as independent risk factors for the prognosis of colorectal cancer.

Description

Technical Field

[0001] This invention relates to the diagnosis of cancer, specifically to the application of novel biomarker compositions in the diagnosis and prognosis of colorectal cancer, and belongs to the field of molecular pathology. Background Technology

[0002] Colorectal cancer (CRC) is one of the most common malignant tumors worldwide. The development of CRC is considered a multi-step process influenced by environmental and genetic factors. CRC primarily occurs in colorectal adenomas, originating from the abnormal proliferation of epithelial cells. Early screening for CRC is crucial for reducing morbidity and mortality. Currently, widely used clinical screening methods for CRC include fecal occult blood tests and serum tumor marker detection such as carcinoembryonic antigen (CEA). However, their sensitivity and specificity are relatively low. Colonoscopy is an effective tool for early screening, but it is painful and invasive for patients, and carries complications such as bleeding, infection, and perforation. Therefore, exploring a simple, non-invasive, effective, sensitive, and specific biomarker is essential for the diagnosis and prognosis of CRC patients and has significant clinical implications.

[0003] Mounting clinical and experimental evidence suggests that tumor-associated systemic inflammation plays a crucial role in the development and progression of cancer-related complications (CRC). Inflammation can promote tumor cell proliferation and angiogenesis. The tumor-associated systemic inflammatory response involves inflammatory mediators and cells. Inflammatory cells release chemicals, particularly reactive oxygen species (ROS), which have oxidative damage and mutagenic effects on tumor cells. To date, most research has focused on peripheral inflammatory cells and their ratios as biomarkers reflecting the overall state of the immune response in cancer patients. These inflammatory markers can be detected through routine complete blood counts, such as neutrophil count, lymphocyte count, monocyte count, platelet count, red blood cell distribution width (RDW), platelet distribution width (PDW), neutrophil-to-lymphocyte ratio (NLR), lymphocyte-to-monocyte ratio (LMR), platelet-to-lymphocyte ratio (PLR), systemic inflammatory response index (SIRI), and systemic immune inflammatory index (SII). These systemic inflammatory markers have demonstrated diagnostic and prognostic value in various cancers, including CRC, gastric cancer, esophageal cancer, lung cancer, breast cancer, pancreatic cancer, and other cancers. Previous studies have found that preoperative NLR and PLR are biomarkers for CRC diagnosis, and NLR combined with CEA can improve the diagnostic efficacy of early CRC. Preoperative NLR and PLR are also significantly associated with the prognosis of colorectal cancer patients. Elevated NLR is associated with poorer survival in colorectal cancer patients. LMR has been used to predict tumor biological behavior, and high LMR is an important indicator of better prognosis in CRC. RDW is a commonly used parameter reflecting erythrocyte size variability and has been used as an indicator of inflammation-related responses. Previous studies have suggested that RDW is associated with the diagnosis, staging, metastasis, and prognosis of CRC. SIRI is defined as monocyte count × neutrophil count / lymphocyte count, and SII is defined as platelet count × neutrophil count / lymphocyte count. In recent years, SIRI and SII have received widespread attention as novel inflammatory biomarkers and are associated with the prognosis of various cancers. However, there are few studies on their clinical value in colorectal cancer.

[0004] To date, most studies have been limited to one or a few inflammatory markers, and the conclusions have been inconsistent. This study systematically and comprehensively analyzes the expression levels of systemic inflammatory markers in patients with colorectal cancer and adenomas, determines their correlation with clinicopathological parameters of colorectal cancer patients, screens valuable systemic inflammatory markers, and analyzes and evaluates their diagnostic and prognostic value for colorectal cancer. Summary of the Invention

[0005] To overcome the limitations of existing technologies and improve the specificity and sensitivity of diagnosis and prognosis of cancer, specifically colorectal cancer, inflammation plays a crucial role in the development and progression of colorectal cancer. This study aims to investigate the expression changes of systemic inflammatory markers during the progression of colorectal cancer and their relationship with clinicopathological parameters, and to evaluate and screen systemic inflammatory biomarkers with diagnostic and prognostic value for colorectal cancer patients.

[0006] The first aspect of this invention is to provide the application of an inflammatory marker in the preparation of cancer diagnostic markers, wherein the inflammatory marker is selected from one or more of NLR, LMR, PLR, SIRI, RDW, RII, and CEA. Preferably, the levels of NLR, PLR, SIRI, SII, and RDW in colorectal cancer patients are significantly higher than those in colorectal cancer adenomas and healthy controls; more preferably, the LMR level is significantly lower than that in the control group and adenoma group. In one specific embodiment, the cancer is colorectal cancer; in another specific embodiment, the inflammatory marker is a composition of SIRI, RDW, and CEA; in yet another specific embodiment, the inflammatory marker is a composition of LMR and RDW.

[0007] The present invention provides another aspect of the application of an inflammatory marker in the preparation of a colorectal adenoma marker, wherein the inflammatory marker is LMR.

[0008] A third aspect of the present invention is to provide an application of an inflammatory marker in the preparation of a cancer prognostic marker, wherein the cancer is colorectal cancer and the marker is RDW.

[0009] Compared with the prior art, the present invention has the following beneficial effects:

[0010] This study is the first to discover that systemic inflammatory markers are associated with colorectal cancer progression. One or more of the following markers—NLR, LMR, PLR, SIRI, RDW, SII, and CEA—have superior diagnostic value compared to other markers, effectively differentiating colorectal cancer patients from healthy individuals and those with adenomas. The combined use of SIRI, RDW, and CEA, as well as the combined use of LMR and RDW, are considered good biomarkers for diagnosing colorectal cancer. Furthermore, RDW and CEA can serve as independent risk factors for CRC prognosis. Attached Figure Description

[0011] Figure 1 : Univariate survival analysis results of RDW in colorectal cancer patients (Kaplan-Meier survival analysis, log-rank test);

[0012] Figure 2Results of univariate survival analysis of CEA in colorectal cancer patients (Kaplan-Meier survival analysis, log-rank test). Detailed Implementation

[0013] The objects and functions of the present invention, as well as the methods for achieving these objects and functions, will be explained below with reference to exemplary embodiments. However, the present invention is not limited to the exemplary embodiments disclosed below; it can be implemented in various forms. This specification is merely intended to help those skilled in the art to comprehensively understand the specific details of the invention.

[0014] Example 1: Discovery of inflammatory markers and their testing in clinical samples

[0015] 1) Research Sample

[0016] The inventors retrospectively analyzed colorectal cancer patients who were first diagnosed and underwent surgery at the Department of General Surgery, Qilu Medical College (Qingdao), Shandong University, between June 2016 and May 2018. None of these patients had received radiotherapy or chemotherapy prior to surgery. All 115 colorectal cancer patients were diagnosed histopathologically; 79 patients with colorectal adenomas from the hospital's health checkup center and 214 healthy controls were also included in the study. Written informed consent was obtained from all patients. Patient data were retrospectively reviewed using the hospital's information database. Tumor staging was performed according to the 2000 WHO Classification of Tumors and the 2002 UICC TNM staging system. Patients with acute or chronic infectious diseases, coronary artery disease, hematological diseases, pulmonary diseases, endocrine or metabolic diseases, and other malignancies were excluded from this study. Postoperative follow-up data were obtained from medical records or telephone interviews, with a median follow-up time of 35.98 months (range 2.14–80.53 months). This study was approved by the hospital's medical ethics committee and complies with the ethical recommendations of the Declaration of Helsinki.

[0017] 2) Detection method

[0018] Peripheral venous blood was collected from healthy individuals and patients with adenomas under basal metabolic conditions; fasting venous blood was collected from colorectal cancer patients within one week prior to surgery and analyzed within two hours after venipuncture. Complete blood count (CBC) was performed using a Sysmex XN 9000 automated blood analyzer (Sysmex Corporation, Japan; reagents were imported directly from Sysmex Corporation). Blood parameters included neutrophil count, lymphocyte count, monocyte count, platelet count, RDW, and PDW. Other systemic inflammatory markers from routine blood tests, including NLR, LMR, PLR, SIRI, and SII, were calculated. Serum CEA was detected using a Roche Cobas e701 immunoassay with electrochemiluminescence immunoassay.

[0019] 3) Statistical analysis

[0020] Statistical analysis was performed using SPSS 20.0 software. Continuous variable data were expressed as mean ± standard deviation or median (interquartile range), and categorical variable data were expressed as frequency. One-way ANOVA, Mann-Whitney U test, and Kruskal-Wallis H test were used for comparisons; chi-square test was used for categorical data. Bonferroni test was used for subgroup comparisons. Receiver operating characteristic (ROC) curves and area under the ROC curve (AUC) were used to evaluate the diagnostic value of systemic inflammatory markers for CRC. Binary logistic regression was used to obtain the pooled predictive value for combined diagnosis of multiple markers; survival curves were constructed using the Kaplan-Meier method, and log-rank test was used for difference analysis. Multivariate survival analysis was performed using a multivariate Cox proportional hazards model; p < 0.05 was considered statistically significant.

[0021] 4) Statistical Results

[0022] a. Changes in the expression of systemic inflammatory markers during colorectal cancer progression

[0023] Inflammation is widely considered to be involved in tumorigenesis and progression. Based on a molecular model of colorectal cancer development, we systematically analyzed the levels of systemic inflammatory markers during the progression from normal mucosa to adenoma to colorectal cancer (as shown in Table 1). The results showed that most inflammatory markers, including neutrophil count, monocyte count, NLR, PLR, SIRI, SII, and RDW, were significantly higher in colorectal cancer patients than in colorectal adenoma patients and healthy controls. Lymphocyte count and LMR levels in colorectal cancer patients were significantly lower than in healthy controls and adenoma groups (p<0.05). However, we found no differences in platelet count and PDW among the three groups. In the adenoma and healthy control groups, only LMR showed a statistically significant difference between the two groups, while other inflammatory markers showed no statistically significant differences.

[0024]

[0025] b. Correlation analysis between systemic inflammatory markers and clinicopathological parameters in colorectal cancer patients

[0026] We further analyzed the correlation between systemic inflammatory markers and clinicopathological parameters in colorectal cancer patients. The results are shown in Table 2. Our data showed that systemic inflammatory markers such as neutrophil count, monocyte count, platelet count, NLR, LMR, SIRI, SII, and RDW were significantly correlated with clinicopathological features associated with advanced tumors, such as tumor size, depth of invasion, and UICC stage (Table 2). However, systemic inflammatory markers were not significantly correlated with patient gender, tumor location, histological grade, or lymph node metastasis.

[0027] We noted that only an increased neutrophil count was associated with increasing age in colorectal cancer patients.

[0028]

[0029]

[0030] c. Analysis of the diagnostic value of systemic inflammatory markers for colorectal cancer

[0031] We further evaluated the diagnostic value of systemic inflammatory markers for colorectal cancer using ROC curve analysis (Table 3). ROC curve analysis showed that LMR had a better diagnostic efficiency than other inflammatory markers. The results showed an AUC of 0.789 [95% confidence interval (CI) = 0.738–0.839]. In addition, RDW and SIRI also showed good diagnostic efficiency, with AUC values ​​of 0.755 and 0.727, respectively. It is generally believed that the diagnostic value of multiple markers combined is higher than that of a single marker. Therefore, we conducted a combined diagnostic analysis of markers with good diagnostic efficiency and found that LMR combined with RDW had the best diagnostic efficiency, with an AUC of 0.835 (95% CI = 0.790–0.881).

[0032] Table 3. AUC values ​​of systemic inflammatory markers for diagnosing colorectal cancer

[0033]

[0034] NLR, neutrophil-to-lymphocyte ratio; LMR, lymphocyte-to-monocyte ratio; PLR, platelet-to-lymphocyte ratio; SIRI, systemic inflammatory response index; SII, systemic immune inflammatory index; PDW, platelet distribution width; RDW, erythrocyte distribution width.

[0035] In addition, we evaluated the potential diagnostic value of systemic inflammatory markers in differentiating colorectal cancer from adenoma (Table 4). The results showed that only LMR (AUC = 0.715, 95% CI = 0.642–0.788), SIRI (AUC = 0.710, 95% CI = 0.638–0.782), and SII (AUC = 0.709, 95% CI = 0.636–0.783) exhibited good diagnostic efficacy. However, combining the three markers did not significantly improve diagnostic efficiency (AUC = 0.760, 95% CI = 0.694–0.826).

[0036] Furthermore, these systemic inflammatory markers were not found to have diagnostic value in distinguishing adenomas from healthy individuals.

[0037] Table 4. AUC values ​​of systemic inflammatory markers for diagnosing colorectal adenomas.

[0038]

[0039] NLR, neutrophil-to-lymphocyte ratio; LMR, lymphocyte-to-monocyte ratio; PLR, platelet-to-lymphocyte ratio; SIRI, systemic inflammatory response index; SII, systemic immune inflammatory index; PDW, platelet distribution width; RDW, erythrocyte distribution width.

[0040] CEA, as a commercially available tumor marker, has been used clinically for the diagnosis and monitoring of colorectal cancer. We used ROC curves to evaluate the diagnostic efficacy of CEA for colorectal cancer (AUC = 0.705, 95% CI = 0.629-0.782). Combining CEA with other inflammatory markers showed that the combined markers were superior to individual markers in diagnosing colorectal cancer, as shown in Table 5. Among these, the combined use of SIRI, RDW, and CEA showed the highest diagnostic efficiency for colorectal cancer, with an AUC of 0.911, and the sensitivity for colorectal cancer identification also increased to 78.3% (Table 6).

[0041] Table 5. AUC values ​​of systemic inflammatory markers combined with CEA in the diagnosis of colorectal cancer.

[0042]

[0043] NLR, neutrophil-to-lymphocyte ratio; LMR, lymphocyte-to-monocyte ratio; PLR, platelet-to-lymphocyte ratio; SIRI, systemic inflammatory response index; SII, systemic immune inflammatory index; PDW, platelet distribution width; RDW, erythrocyte distribution width.

[0044] Table 6. Sensitivity and specificity of SIRI, RDW, and CEA in diagnosing colorectal cancer.

[0045]

[0046] d. The prognostic value of systemic inflammatory markers in colorectal cancer patients

[0047] Kaplan-Meier survival analysis and log-rank test were used to evaluate the prognostic value of clinicopathological parameters in colorectal cancer patients. Results showed that patient age, tumor invasion depth, lymph node metastasis, and UICC stage were statistically correlated with prognosis.

[0048] To evaluate the prognostic value of systemic inflammatory markers, optimal cutoff values ​​were determined based on ROC curves. All cases were divided into high-value and low-value groups according to the cutoff values ​​of systemic inflammatory markers. Kaplan-Meier cumulative survival curves showed a significant difference in RDW (p = 0.024). Figure 1 CEA, as a tumor marker for CRC, has a normal clinical reference range of 0-5 ug / L. We also found that patients with elevated CEA levels had a poorer prognosis (p = 0.016). Figure 2 ).

[0049] Univariate Cox proportional hazards models showed that overall survival in colorectal cancer patients was statistically correlated with age, tumor depth, lymph node metastasis, CEA, and RDW (Table 7). All important indicators affecting patient survival were included in a multivariate Cox proportional hazards model, with histological grade, tumor size, UICC grade, NLR, LMR, SIRI, SII, PLR, and PDW also included in the model. The final results showed that independent prognostic indicators included age (p<0.001), UICC stage (p=0.001), CEA (p=0.030), and RDW (p=0.022). Only the inflammatory marker RDW served as an independent risk factor for CRC prognosis (Table 7).

[0050] Table 7. Analysis of prognostic factors in colorectal cancer patients using univariate and multivariate Cox proportional hazards models.

[0051]

[0052] NLR, neutrophil-to-lymphocyte ratio; LMR, lymphocyte-to-monocyte ratio; PLR, platelet-to-lymphocyte ratio; SIRI, systemic inflammatory response index; SII, systemic immune inflammatory index; PDW, platelet distribution width; RDW, erythrocyte distribution width.

Claims

1. Application of inflammatory marker combination detection reagents in the preparation of colorectal cancer diagnostic reagents, wherein, The combination of inflammatory markers is SIRI, RDW, and CEA.

2. The application according to claim 1, wherein, The levels of SIRI and RDW were significantly higher in patients with colorectal cancer than in patients with colorectal adenomas and healthy controls.

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