The prognostic value of CA19-9, D-dimer and expression of TNFAIP3(A20) protein in pancreatic ductal adenocarcinoma patients

Background: To explore signi�cance of CA19-9, D-dimer with TNFAIP3 (A20) protein and evaluate its prognostic signi ﬁ cance in patients suffering from pancreatic ductal adenocarcinoma (PDAC). Methods: 148 patients suffering from pancreatic ductal adenocarcinoma in Northern Jiangsu People’s Hospital a�liated to Yangzhou University between January 2012 to December 2016 were studied. Cutoff values of prognostic factors were predicted by Receiver operating characteristic curve (ROC curve). Kaplan-Meier method was used to describe survival curve of patients. Univariate and multivariate regression analyses were used to analyze prognostic factors of patients. Results: The recommended cutoff values of the neutrophil-lymphocyte rate (NLR), platelet-lymphocyte rate (PLR), CA19-9 and D-dimer were 2.04 (sensitivity, 0.59; speci�city, 0.9; area under the ROC curve (AUC), 0.749; P<0.001), 52.94 (sensitivity, 0.73; speci�city, 0.95; AUC, 0.829; P<0.001), 176.66U/ml (sensitivity, 0.7; speci�city, 0.9; AUC, 0.794; P<0.001) and 1.18mg/L (sensitivity, 0.82; speci�city, 0.9; AUC, 0.845; P<0.001) respectively. The expression of CA19-9 in serum was related with lymph node metastasis (P = 0.010), tumor lymph node metastasis (TNM) stage (P <0.001) and survival ratio (P <0.001). The D-dimer was positively related with differentiation grade (P=0.014), tumor size (P=0.045), TNM stage (P=0.006) and survival ratio (P<0.001). A20 was positively related with differentiation grade (P<0.001), BMI (P<0.001), TNM stage (P=0.024) and survival ratio (P<0.001). The Kaplan-Meier curves showed that the patients with pancreatic ductal adenocarcinoma had a signi�cant difference in the expression of CA19-9 group, expression of D-dimer group and expression of A20 (P<0.05). CA19-9, D-dimer, TNM stage, differentiation grade and A20 were independent prognostic markers for patients sufferingfrom pancreatic ductal adenocarcinoma by univariate and COX multivariate analyses. Conclusions: CA19-9, D-dimer and A20 were independent prognostic markers for pancreatic ductal adenocarcinoma patients.


Background
Patients with pancreatic cancer, as one of the most lethal malignant tumors, were usually found to metastasize to distant organs.The possible reasons may be no speci c symptoms in the early stage of pancreatic cancer [1,2].Although many therapies were used to treat pancreatic cancer, 5-year survival rate was less than 9% in patients with pancreatic cancer [3,4].The prognosis of pancreatic cancer was closely associated with early detection and treatment.Now the main diagnostic method of pancreatic cancer was through imaging technology or pancreatic biopsy.Researchers had begun to discover prognostic blood markers, containing the neutrophil-lymphocyte rate(NLR) [5,6] and platelet-lymphocyte rate (PLR) [7].
CA19-9 and D-dimer played important roles in the survival of pancreatic cancer [8,9].CA19-9 was a sialic acid containing glycan antigen found in both glycoproteins and glycolipids and used as a marker of pancreatic cancer or other tumors [10].D-dimer was a soluble brin degradation product that resulted from ordered breakdown of thrombi by the brinolytic system.The results of this meta-analysis indicated that plasma D-dimer levels can be used as an important reference for the early identi cation and staging of breast cancer [11].D-dimer was reported as an independent prognostic marker for some cancer [12,13].A20 was a cytoplasmic zinc-nger protein, which was found to be expressed in a variety of human cells, which associated with disease [14].At present, A20 was reported to be associated with in ammatory responses by regulating canonical NF-κβ pathway, which played key roles in termination of the active canonical NF-κβ pathway [15].The study suggested that miR-125a promoted chemo-resistance to gemcitabine in pancreatic cells through targeting A20 [16] and A20 expression was reduced in pancreatic cancer tissues and signi cantly associated with the pancreatic cancer behavior [17].
Although CA19-9 is a marker of pancreatic tumor, It is easily interfered by other factors, such as jaundice [18]and in ammatory level [19].D-dimer and A20 have also been studied in cancer [12,17].As a stress response gene in endothelial cells (ECs), A20 has a protective effect from tumor necrosis factor (TNF)-mediated apoptosis and inhibits in ammationis [20,21].D dimer level is associated with vascular endothelial cell injury [22].Therefore, A20 may be associated with D-dimer and CA199.The prognostic value of CA19-9, D-dimer and A20 in patients with pancreatic ductal adenocarcinoma have not been thoroughly and extensively discussed.In this study, three indicators were selected to explore the relationship between evaluation and survival of pancreatic cancer in patients with pancreatic ductal adenocarcinoma.

Inclusion and exclusion criteria
The study was approved by the Ethics Committee in Northern Jiangsu People's Hospital a liated to Yangzhou University.All patients had signed the wrirren informed consent.This study got carried out according to the guidance from Statement of Helsink.
Inclusion criteria included: 1)Patients was diagnosed as pancreatic ductal adenocarcinoma by pathological diagnosis; 2)Patients did not receive radiotherapy and/or adjuvant chemotherapy before the resection; 3)Patients were determined to require surgical treatment; 4)The patient's peripheral blood tests were performed within one week before operation.
Exclusion criteria included 1) Patients previously had malignant tumors or other primary tumors; 2) Patients suffered from certain diseases that affected peripheral blood cell count, such as infection; 3) Patients had accepted radiotherapy and/or adjuvant chemotherapy previously before operation; 4) Patients died within four weeks after the operation.

Patient characteristics and tissue specimens
148 patients who were diagnosed as pancreatic ductal adenocarcinoma and subjected to curative surgery during the Northern Jiangsu People's Hospital a liated to Yangzhou University from January 2012 to December 2016, were studied.the patients that were being investigated in this study are all resectable.
Information on the basic characteristics of the patient was collected (Table 1), including blood tests and pathological features.The progression of the pancreatic cancer patients were classi ed according to the eighth vision of American joint committe on cancer staging system.The patient's peripheral blood tests were performed within one week before operation.Prognostic Value of NLR and PLR in Patients with pancreatic ductal adenocarcinoma were analyzed.The recommended cutoff values of CA19-9, D-dimer, PLR and NLR before operation were determined by ROC curve based on the Youden nger (maximum (sensitivity + speci city-1)).

Immunohistochemical analysis
According to standard procedures of immunohistochemical procedures, the tissues of 148 patients with pathologically diagnosed pancreatic ductal adenocarcinoma were xed in formalin and the tissues were embedded in para n.The para n tissue was cut into 4 µm thick sections, and the sections were dewaxed and hydrated.Rinse sections with 10 µmol/L citrate buffer and heat under the microwave for 10 minutes.The sections were then immersed in 3% hydrogen peroxide and anhydrous methanol.Sections were treated with 10% goat serum albumin for 20 minutes.Add a primary antibody (rabbit polyclonal antibody, Abcam, USA) to A20 protein diluted 1: 100 to the section, store it at 4 ℃ overnight, and then add a secondary antibody at room temperature, Incubating for 1 hour.The sections were incubated with horseradish peroxidase-conjugated streptavidin for 1 hour, and the sections were developed with diaminobenzidine (DAB).Finally, the sections were counterstained with Mayer's hematoxylin.

Follow-up and treatment
Our study collected information of 162 patients with pancreatic ductal adenocarcinoma.After operation, some patients with poor health were given supportive therapy, and some patients received chemotherapy or targeted treatment.Their follow-up data were obtained by telephone and outpatient service.The registered patients were followed up every month.All the patients were followed up, eight people lost contact and six patients died from non-cancer diseases, who were excluded from the study.148 patients with pancreatic ductal adenocarcinoma were included in this study.The follow-up period of our study ended in December 2018.

Statistical explanation
Statistical analysis was analyzed by using SPSS 23.0 software (SPSS Inc., Chicago, IL).The cutoff values of indexes was performed according to the ROC curve based on the Youden nger (maximum (sensitivity + speci city-1)).Categorical variables were analyzed by Pearson x 2 test or Fisher's exact test.
For overall surviving, Kaplan Meier curves got established using the log-rank test.the Cox appropriate hazard pattern was used for survival analysis.P < 0.05 was de ned as statistically signi cant.

Discussion
It is known that pancreatic ductal adenocarcinoma is one of the high malignant degree of cancer.At present, several biomarkers for pancreatic cancer have been proposed for earlier diagnosis, there is no effective diagnostic method to nd early pancreatic in clinical application cancer, surgical resection offers the chance to cure PDAC.But Most of PDAC patients have lost the opportunity of operation when pancreatic cancer was found.To found biomarkers for early diagnosis of pancreatic cancer propose future research directions [23][24][25].
Surgery is one of the most important therapy for pancreatic ductal adenocarcinoma.However, due to the limitation of diagnostic techniques, it is usually di cult to detect pancreatic cancer early, which leads to poor prognosis [24].Some scholars had shown that some markers may be related to the prognosis of PDAC patients, for examples, differentiation grade and TNM stage [1,8].These prognostic factors were di cult to judge preoperatively, so the study of preoperative serum markers had become a research hot spot [12,26].As an independent prognostic indicator, CA19-9 had attracted more and more attention [26].Level D-dimer was found to be associated with prognosis of digestive tumors [12,13].But CA19-9 level may be affected by jaundice [18]and in ammatory [19], many pancreatic cancer patients complicated with jaundice.
In our study, patients with preoperative CA19-9 ≥ 176.66 U/ml had a high degree of malignancy and a short overall survival period, but the mechanism of CA19-9 and tumor progression was still unclear.CA19-9 was positively correlated with lymph node metastasis (P = 0.010), TNM stage (P < 0.001), survival ratio (P < 0.001).D-dimer degraded by plasmin during brinolysis was a degradation product of the crosslinked brinpolymer.Previous studies had showed that plasma level of D-dimer can predict poor prognosis in several types of malignant tumors, including ovarian [27], breast[28] and colorectal [29].The D-dimer was positively correlated with differentiation grade (P = 0.014), tumor size (P = 0.045), TNM stage (P = 0.006) and survival ratio (P < 0.001).Apart from being a diagnostic marker for activation of coagulation and brinolysis, D-dimer also played an important role in cancer progression, invasion, and prognosis [29].Recent research showed that activation of the coagulation system was associated with tumorigenesis development, dissemination and transfer [31,32].Fibrinolytic enzymes in brinolytic systems played an important role in tumor invasion and penetration into the blood circulation.Studies had also shown that tumors were encapsulated in the network structure, as the disease developed and the rate of metastasis accelerated, the release of D-dimer increased after the destruction of the network structure, and thrombotic diseases were prone to occur [12,33,34].
A20 was signi cantly correlated with differentiation grade (P < 0.001), BMI (P < 0.001), TNM stage (P = 0.024) and survival ratio (P < 0.001).As a cytoplasmic zinc-nger protein, A20 was reported to be expressed in a variety of human cells, such as T lymphocytes and B lymphocytes [35,36], which had been reported to regulate dynamic immune responses by negatively mediating the activity of the transcription factor NF-κβ and the proin ammatory gene expression [14,37,38].NF-κβ signaling pathways involved in regulating tumor proliferation and apoptosis.A20 participates in endothelial cell stress response and in ammatory response [15,21], which may be associated with D-dimer and CA19-9.Their mechanism is still unclear, which is also our next research direction.A20 in PDAC was low expression, which found to be linked with TMN stage (P < 0.05).Other researchers suggested that expression of A20 was reduced in pancreatic cancer tissues, but not deeply associated with TNM stages [17].The overall survival time of patients with CA19-9 < 176.66 U/ml (P < 0.001), D-dimer < 1.18 mg/L (P < 0.001) and high expression of A20 group (P < 0.001) was longer by Kaplan-Meier curves.
TNM stage and differentiation grade were not easy to be evaluated by imaging examination before operation, however, the levels of preoperative CA199 and D-dimer was easier to be detected, which may be helpful to evaluate the prognosis of pancreatic ductal adenocarcinoma patients.For patients with high levels of CA19-9 and D-dimer before operation, early intervention can be considered.We can not only use the CA19-9 and D-dimer level to judge the prognosis, but also A20, TNM stage and differentiation grade should also be considered comprehensively after the surgery.
We should make su cient evaluation and preparation for the patients with PDAC before operation, and make the best individualized treatment plan for the patients.Follow-up should be carried out after operation to nd early recurrence or metastasis to prolong survival time of patients.Therefore, by evaluation of relevant indexes before and after surgery, the optimal comprehensive treatment plan for PDAC patients was formulated, which may improve the prognosis of patients, which required a lot of further clinical and basic research.

Table 3
Proportional risk model for predicting overall survival Yao Jie and A-Ma Xu carried out the main work and contributed equally.They designed this study, drafted this manuscript and revised this work.XiaoDong Wang, ZhengNan Li and JianJun Qian performed the study and participated in this work.All authors read and approved the nal manuscript.Department of general Surgery, the fourth A liated Hospital of Anhui Medical University, Hefei, 230000, China; 2 Department of Hepatobiliary and Pancreatic Surgery, Northern Jiangsu People's Hospital a liated to Yangzhou University, Yangzhou, 225001, China; 3 Department of general Surgery, the rst A liated Hospital of Anhui Medical University, Hefei, 230000, China.