ZENG, Kai; LIU, Qi-cai; LIN, Jian-hua; LIN, Xin-hua; ZHUANG, Ze-hao; GAO, Feng; OU, Qi-shui

Pancreatic cancer is an aggressive disease with a poor prognosis. Hereditary factors have been reported in up to 10% of the pancreatic cancer cases.^1–3 The mutations of genes such as K-ras, palladin and BRCA2 have been found in patients with pancreatic cancer.⁴ In recent years, the gene location and mechanism of the hereditary pancreatitis are in-depth study which are mostly caused by the mutations of cationic trypsinogen (PRSS1) gene which will increase 53 times to incidence of pancreatic cancer.⁵ So there is a growing recognition that PRSS1 gene disorder is related to pancreatic cancer and studying the different genotypes of PRSS1 may provide some clues to the genetic susceptibility of pancreatic cancer.

PRSS1 gene mutations commonly exist in patients with hereditary pancreatitis and 40% of whom will develop to pancreatic cancer.⁶PRSS1 gene is not an oncogene, nor a tumor-suppressor gene, nor a genome maintenance gene. Although it is not a cellular controller, it plays an important role in reparing and self-stability maintaining of cell or tissue. If something happened to the gene, like missing or weakening in function, it will result in random mutation in DNA replication and accumulation that significantly increases the risk of pancreatic ductal adenocarcinoma. Furthermore, trypsinogen is also related to a wide variety of tumors’ process.⁷ So, it is necessary to study the relationship between the PRSS1 genotypes and pancreatic cancer.

METHODS

Subjects

A total of 156 patients with pancreatic cancer and 220 unrelated individuals with an ethnic distribution comparable to the patients as normal controls were under investigation. All of the patients were Han population diagnosed by pathology.

DNA analysis

Genomic DNA was extracted from peripheral blood specimens and pancreatic cancer tissue using a QIAamp DNA mini kit (Qiagen, Germany). The entire coding sequence of the PRSS1 gene was examined by polymerase chain reaction (PCR) and direct sequencing of the coding exons 1 to 5 was done. Primer pairs3 and experimental conditions used to generate four fragments were as follow: the 50 μl reaction mixture contained 200 ng of genomic DNA, 10 mmol/L Tris HCl (pH 9.0), 50 mmol/L KCl, 0.1% Triton, 2 mmol/L MgCl2, 0.25 mmol/L dNTPs, 100 ng of sense primer, 100 ng of antisense primer and 3.0 U Taq-DNA polymerase. Different primers were separated in different PCR tubes after amplification under the same conditions. Cycling conditions included an initial step at 94°C for 5 minutes, then 34 cycles at 94°C for 1 minute, 52°C for 1 minute, 72°C for 1 minute with a final elongation step at 72°C for 7 minutes. The PCR products were purified for sequencing after electrophoresis on an agarose gel. For sequencing, a Perkin Elmer Big Dye Sequencing kit (Perkin-Elmer, Shelton, CT, USA) and an ABI PRISM7700 sequencer (Perkin-Elmer ABI, Foster City, CA, USA) were used. MutectorTM KRAS kit (TrimGen Genetic Diagnostics, USA) was used to identify k-ras gene mutation in codons 12 and 13. If the common mutations of k-ras gene screening were negative, the whole k-ras gene was then directly sequenced to confirm k-ras gene undisorder.

Ethics

Informed consents were obtained from all patients before the study.

RESULTS

Two patients brought IVS 3 +157 G>C mutation of PRSS1 gene (Figure 1), neither of them showed any symptoms until they developed jaundice. A novel complicated mutation (c.409 A>G and c.416 C>T) was found in a patient with young onset pancreatic cancer (Figure 2). The complicated mutation leads to No.135 and No.137 amino acids change, which are Thr→Ala and Thr→Met mutations in trypsinogen where the point of integrating pancreatic secretory trypsin inhibitor (PSTI) is. According to the data of National Center for Biotechnology Information (NCBI), we made the conclusion that IVS 3 +157 C>G and c.409 A>G were novel mutations while c.416 C>T had been reported in a 13-year-old Asian girl with acute pancreatitis by Keiles and Kammesheidt8 in 2006. K-ras gene mutation was not found in peripheral blood or pancreatic tissue in the three patients with PRSS1 gene mutation. The dramatic discovery of these cases drived us to study deeper with tremendous interest.

Figure 1.

Figure 2.

The younger patient was 28 years old, admitted to our hospital with a history of jaundice and steatorrhea. Biochemical test showed that he got diabetes mellitus. Diagnostic imaging demonstrated a pancreatic pseudocyst at the head of pancreas. Fine-needle aspiration pathology showed developed adenocarcinoma. After a comprehensive assessment, the patient was considered to have chemotherapy. Other laboratory test results: antinuclear antibody profile R0–52 (+), liver autoantibodies (-), keratin antibody (-), anti-neutrophil cytoplasmic antibodies (ANCA) (-). Serum hyaluronic acid and collagen IV of this patient was significantly higher than other patients.

DISCUSSION

The incidence of pancreatic cancer has been increasing in recent years which threats human health seriously.^1,3–9 What's more, individual's susceptibility to the tumor is closely related to the genetic background.^10–12 This study took a series of investigations into the critical pathogenic gene of pancreatic cancer — PRSS1 gene and took the lead in clarifying the mutation feature of PRSS1 gene in patients with pancreatic cancer among Han population.

In the last years, many studies show that trypsin plays an important role in physiological functions and physiological processes such as digestion, coagulation function, embryonic development, et al.^11,12 Once the trypsinogen was activated, lots of proenzymes would turn into isozyme which leads to cell response. That is to say, trypsinogen plays an important role in maintaining environmental balance, if its function or quantity is destroyed; the immune system may lose its balance and can not detect the mutational cell.

K-ras gene mutation is widely recognized as an associated factor of pancreatic cancer, the mutation can be detected as high as 80%-90% in pancreatic cancer patients according to literatures.¹³ But the test requires surgical puncture of the pancreatic tissue, which limits its clinical applicability as a reasonable method in early screening for pancreatic cancer. Unlike k-ras gene, PRSS1 gene mutation can be found in peripheral blood specimens.^14,15 The young patient was unusually found with p.T135A and p.T137M mutations. And there were not any disorders of k-ras gene detected in the cancer tissue. It may be that the p.T135A/ p.T137M mutation which occurs at the binding site of trypsinogen inhibitor PSTI leads to abnormal activation of trypsin. The abnormal expression of the trypsin results in a series of hydrolysis process because of related precursor proteins’ activation or blocked inhibition that leads to the body environmental damage and gives rise to tumors.¹⁶

At first, PRSS1 gene mutation was found in hereditary pancreatitis and the risk of pancreatic cancer is more than 50 times than the normal. About 40% of the patients with PRSS1 gene mutations will develop into pancreatic ductal adenocarcinoma. The finding of p.T137M mutation is a good example to illustrate the link between pancreatitis and pancreatic cancer. It also disclosed the idea that pancreatic cancer comes from chronic inflammation of the pancreas not from an oncogene. Of course, these findings, if confirmed by any large-scale population surveys, may provide opportunities for discovering novel markers that can help in diagnosis for pancreatic cancer and in predicting a patient's tolerance to hereditary susceptibity and overall clinical outcome.

REFERENCES