Department of 1Pathology, The First Affiliated Hospital, 2Pharmaceutical Analysis, and 3Bioinformatics, Fujian Medical University, Fuzhou 350005, People’s Republic of China
4Pharmaceutical and Medical Technology College, Putian University, Putian 351100, China
Received April 29th, 2017 - Accepted June 12th, 2017
Background Pancreatic cancer is a rapidly progressive and usually fatal disorder that has risen to be the 6th leading cause of cancer deaths in China. Trypsin is believed to play an important role in the development of pancreatic cancer. The aim of the present study was to determine whether serum trypsin levels and cationic trypsinogen (PRSS1) genotypes could be served as prognostic indicators in pancreatic cancer. Methods A total of 140 patients with pancreatic cancer were included in this study. The serum trypsin levels were determined by enzyme linked immunosorbent assay. The genotypes of PRSS1 gene were analyzed by polymerase chain reaction -direct sequencing. And the clinicopathologic parameters of patients were also evaluated. Results It showed that the median survivals of patients with high serum trypsin level (6.4 months) were significantly shorter than that who with low serum trypsin level (7.6 months; P=0.0192). The median survivals were also found significantly shorter in patients who had rs10273639 CC genotype of PRSS1 (4.1 months) compared to those who had TT (8.4 months) or TC (6.6 months) genotypes (P=0.0058 for TT vs. CC, P=0.0377 for TC vs. CC). However, there were no significant differences in the median survivals between patients with TT and TC genotypes of PRSS1. Conclusions Serum trypsin levels and the rs10273639 CC genotype of PRSS1 may be used as novel prognostic indicators in pancreatic cancer.
Biomarkers; Pancreatic Neoplasms; Prognosis; PRSS1 protein, human; Trypsin
Pancreatic cancer is responsible for one of the most lifethreatening malignancies all over the world . Although the progress of surgical and intensive inspect techniques, the outcome of pancreatic cancer remains disappointing and the overall 5-year survival rate is only 6% . And it has risen to be the 6th leading cause of cancer deaths in China . The poor prognosis is mainly attributed to late diagnosis, early local invasiveness and distant organs metastasis . And the exact molecular mechanisms underlying pancreatic cancer remain to be clarified . Therefore, it is urgent to find novel useful diagnosis and prognostic biomarkers for early diagnosis and effective treatment of pancreatic cancer .
The abnormal activation of trypsinogen may cause an imbalance between enzymes and antienzymes, which may result in inflammatory injury and microenvironment damage in the pancreas . These changes contribute to the development of pancreatitis and cell carcinogensis [7, 8]. Trypsin can act as a signaling factor to promote the proliferation of cancer cells, and digest the matrix to accelerate the invasion and metastasis of cancer cells [9, 10]. Moreover, trypsin may also serve as a stimulator of lymphocytes , and play a vital role in the immune tolerance for mutated cells, which provides a selective advantage environment for the growth of cancer cells [12, 13, 14]. Therefore, the abnormal of trypsin is close related to pancreatic cancer, and may be an early diagnostic or prognostic maker for pancreatic cancer. Several studies have found that the prognosis is positively associated with the genetic background of patients [15, 16, 17]. Our previous study found that different rs10273639 genotypes in the promoter of cationic trypsinogen (PRSS1) gene could affect the quantity of trypsin , which lead to the abnormal activation of PAR-2 and subsequently changed the distribution of cell cycle and induced the onset of pancreatic cancer [19, 20]. Our previous results showed that rs10273639 genotypes of PRSS1 gene in peripheral blood are positively correlated with the risk of pancreatic cancer . Researchers also found that the prognosis of hereditary pancreatitis could be poor with the presence of PRSS1 mutations [22, 23]. To date, most of the studies showed that the mutations of PRSS1 were related the onset of hereditary pancreatitis . However, the relation between PRSS1 variations and the prognosis of pancreatic cancer has not yet been well established.
In this study, we investigated the serum trypsin levels and rs10273639 genotypes of PRSS1 in patients with pancreatic cancer, assessed their correlations with the clinicopathologic parameters as well as the survivals of patients.
A total of 140 patients with pancreatic cancer who had not undergone any form of antitumor therapy prior to admission at the 1st Affiliated Hospital of Fujian Medical University between May 2013 and January 2016 were consecutively enrolled in our study. The diagnosis of pancreatic cancer was based on the 7th edition of the American Joint Committee on Cancer (AJCC) staging system . Patients who did not satisfy with the indication for operation, or did not undergo surgical treatment were excluded from the study. For the purpose of this study, the clinic-pathological parameters including age, sex, histological information laboratory parameters were also collected. Venous blood samples were obtained before surgery from each patient at the time of diagnosis for detecting trypsin level and PRSS1 genotyping. Sera were separated immediately and stored at −80°C until trypsin enzyme-linked immunosorbent assay was done. DNA was isolated from blood samples and was kept at −20°C. The study protocol was approved by the Ethics Committee of Fujian Medical University. Written informed consents were obtained from all the patients.
Serum Trypsin Measurement
The serum trypsin was tested with ELISA kits (Human trypsin ELISA Kit; Ameko, Shanghai, China) according to the manufacturer’s protocol.
DNA Isolation and PRSS1 Genotyping
Genomic DNA was isolated from blood sample using TIANamp Blood DNA Kit (Tiangen Biotect, PR China). PRSS1 genotypes were detected using PCR-direct sequencing, as previously described .
Statistical analysis was performed using the SPSS 18.0 software package (SPSS Inc., Chicago, IL, USA). Normal variables were expressed as mean ± SD and compared by ANOVA test. Abnormal continuous variables were expressed as median (min-max) and compared by the Kruskal - Wallis H test. Comparisons were made using χ2 test for categorical variables. Survival data were analyzed by using Kaplan-Meier survival curves and log-rank test. For all tests, a two-sided P value <0.05 was considered to be statistically significant.
A total of 140 patients with pancreatic cancer were recruited into this study. Their clinicopathological characteristics including age, survival and pathological types were summarized in Table 1.
The relationship between PRSS1 genotypes and clinicopathologic features in pancreatic cancer: There were three PRSS1 genotypes of rs10273639 (TT, TC and CC). The correlation between these genotypes and clinicopathological features in pancreatic cancer was illustrated in Table 2. We found the PRSS1 genotype was obviously associated with the length of survival (P=0.043). However, the other clinical factors, such as age, pathological types, serum trypsin levels, liver functions, serum tumor marker levels and lipid levels, suggested no significant association with PRSS1 genotypes. In order to further investigate the correlation between PRSS1 genotypes and the prognoses of pancreatic cancer, Kaplan- Meier analysis and log-rank test were performed to analyze the association between PRSS1 genotypes and 140 patients' survival. Patients with rs10273639 CC genotype had a significantly shorter overall survival than who with TT (P=0.0058) and TC genotype (P=0.0377) (Figure 1), though there were no obvious differences between TT and TC carriers. These findings implicated that CC genotype of PRSS1 gene is positive with poor outcome in pancreatic.
Figure 1. The relation between PRSS1 genotypes and survival. Kaplan- Meier analysis and log-rank test were performed to analyze the association between PRSS1 genotypes and 140 patients' survival. Patients with rs10273639 CC genotype had a significantly shorter overall survival than who with TT (P = 0.0058) and TC genotype (P = 0.0377).
The relationship between serum trypsin and clinicopathologic features in pancreatic cancer: In order to evaluate the relationship between serum trypsin concentration and clinicopathological characteristics in pancreatic cancer, the patients were divided into low serum concentration (≤16.0 ng/mL) and high serum concentration (>16.0 ng/mL) groups, according to the mediun serum concentration of all the patients. As shown in Table 3, the serum glucose in low serum trypsin group was significant higher compared to high serum trypsin group (P=0.029). Importantly, Kaplan-Meier analysis revealed that patients with high serum trypsin concentration had a significantly worsened survival (6.4 months) than who with low serum trypsin concentration (7.6 months) (P=0.0192) (Figure 2), which reflects that high serum trypsin concentration could be a valuable a prognostic indicator for pancreatic cancer. But no statistically significant differences were found in other clinical factors between the two groups.
Figure 2. The relation between serum trypsin concentration and survival.
Kaplan-Meier survival analysis of 140 patients with PC based on serum
According to the medium of serum trypsin concentration, patients were separated into low serum concentration (≤16 .0ng/ml) and high serum concentration (＞16.0 ng/ml) groups. Patients in the high serum trypsin concentration group had a significantly shorter overall survival than who in the low serum trypsin concentration group (P = 0.0192).
This study was conducted to explore the relationship of trypsin and PRSS1 genotypes with various clinical and pathological parameters as well as the prognosis of pancreatic cancer. Our findings showed that high serum trypsin level and rs10273639 CC genotype of PRSS1 were positive related to the short survival of pancreatic cancer, which inflects that serum trypsin levels and the CC genotype of PRSS1 may serve as novel prognostic indicators for pancreatic cancer.
Our previous study had demonstrated that the medium serum trypsin concentration in patients with pancreatic cancer was 1.68 times higher than that in healthy controls . High serum trypsin not only causes an imbalance between enzymes and antienzymes, which may result in inflammatory injury and microenvironment damage in the pancreas  but also serves as a risk factor for pancreatic cancer, inducing pancreatic cells malignant transformation through proteinase-activated receptor-2 (PAR-2)- p-ERKI/2- proliferating cell nuclear antigen (PCNA) signal pathway [26, 27, 28]. Moreover, trypsin may digest the extracellular matrix and basement membranes, and contribute to the invasion and migration of pancreatic cancer cells [29, 30, 31]. Therefore, the abnormal of trypsin may be related to the outcome of pancreatic cancer. Our results showed that high serum trypsin level was positive related to the short survival of pancreatic cancer, which is not only well in line with these theories, but also indicates that serum trypsin may be a useful prognostic maker for pancreatic cancer.
Serum trypsin levels may be affected by several factors including PRSS1 variations. Our previous studies found that trypsin was highly expressed in the pancreatic tissues in patients with PRSS1 mutation  and was associated with the rs10273639 genotypes in neonates with sepsis . These studies showed that the genetic variations in the PRSS1 gene may increase the transcriptional activity of promoters or enhance the stability of abnormal trypsin to increase the quantity of trypsin. But in this study, we didn’t find any differences among rs10273639 TT, TC and CC genotypes. This case may be caused by different subjects and diseases. As for pancreatic cancer, the pancreatic microenvironment, especially the pancreatic cells damage, may also trigger the release of more trypsin. In this study, we found that the serum glucose in low serum trypsin group was significant higher compared to high serum trypsin group, which may also reflect that the trypin levels positively related to the pancreatic microenvironment change. Additionally, we also found that patients with rs10273639 CC genotype had a significantly shorter survival than whom with TT and TC genotypes. Our results implicated that the CC genotype of PRSS1 was positive with poor outcome in pancreatic cancer and may be act as a prognostic maker for pancreatic cancer.
Our results demonstrated that high serum trypsin level and the CC genotype of PRSS1 were positive related to the short survival of patients with pancreatic cancer. Thus, high serum trypsin level and rs10273639 CC genotype of PRSS1 may serve as novel prognostic indicators for pancreatic cancer.
This work was supported by a grant from the National Natural Science Foundation of China (no.81571613, no.8152442) and Joint Fund for Program of Science innovation of Fujian Province (No.2016Y9011). The authors also thank Dr. Qingquan Chen, Fujian Medical University, China, for his help with formatting the manuscript.
The authors declare that they have no conflict of interest.
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