Study On Detecting Hypermethylation Of The Related Genes In Pancreastic Juice To Diagnose Pancreatic Diseases

Study on detecting hypermethylation of the related genes in pancreastic juice to diagnose pancreatic diseasesINTRODUCTIONPancreatic cancer (PCa) is still one of the most common malignant cancers to human, and it is the fourth and sixth cause of death in American and Europe, and also the fifth cause of death in Japan, in our country is the tenth cause of death. 90% of patients with pancreatic carcinoma will die in one year after diagnosis, and the average survival time after diagnosing is three months. Patients suit for surgical operation is less than 15%, and 5 years survival rate is less than 4% after resecting tumor by surgical operation. Different from gastrointestinal tumor, it is difficult to biopsy for its anatomical character. Despite efforts in the past years, conventional treatment approaches, such as surgery, radiation, chemotherapy, or combinations of these, have little impact on the course of this aggressive neoplasm. Therefore, only by developing a detailed understanding of the molecular biology of pancreatic cancer will we be in a position to effectively diagnose, prevent, and treat this disease.The genesis of human cancers, including those of the pancreas, is regarded as a multistep process involving cumulative genetic and epigenetic alterations of tumor suppressor genes. On the other hand, epigenetic alterations including hypermethylation in DNA and modification in histone of gene promoters are proving to be consistent and early events in varied neoplastic progression. Such alterations are thought to contribute to inactivation in many types of cancer. In PCa, several cancer-related genes, including p16, RASSFlA, 3-OST-2, cyclin D2, SOCS-1, RAR-β, APC, and others, undergo aberrant methylation during PCa development and many of these genes is rarely detected in non-neoplastic pancreatic tissues. Many of these genes are aberrantly methylated in a high proportion of PCa and can be detected with methylation specific polymerase chain reaction (MSP) make them potentially attractive for early detection. Pure pancreatic juice(PPJ) is thought to have a high concentration of DNA and proteins released from PCa compared with other clinical sources such as serum, and can be collected easily by ERCP(endoscopic retrograde cholangiopancreatography). Recently, intitial studies had been performed to detect aberrantly methylated genes in the pancreatic juice from patients with PCa as a promising diagnostic strategy. To further evaluate the diagnostic significance of detecting aberrant methylation in PPJ for diagnosis of PCa, we investigate aberrant methylation of neuronal pentraxinâ…¡(NPTX2), claudin 5 (CLDN5), and Preproenkephalin(ppENK) genes which were methylated in over 90% of primary pancreatic cancer tissues and rarely methylated in normal pancreatic ductal epithelia, using MSP and realtime MSP methods.MATERIALS AND METHODSâ… Culture of pancreatic carcinoma cell lineThe 9 human Pca cell lines, KLM1, MiaPaCa2, Panc1, PK45H, PK59, PK1, PK8, PK9 and BxPC3 were used for this study. They were cultured in PRMI1640 and DMEM with 10% fetal calf serum in the condition of 37℃, 5%CO₂. those cell lines were provided by Department of Internal Medicine at Kanazawa University Cancer Research Institute Hospital and affiliated hospitals.â…¡Patients and pancreatic juice samples collection.We studied 80 patients who were diagnosed and treated at the Department of Internal Medicine of the Cancer Research Institute Hospital at Kanazawa University and the Knazawa University Hospital and its Affiliated medical facilities in Japan from 1995 to 2005. Informed consent for this study was obtained from all the patients. Ultrasound Sonogramphy(US), computed tomogramphy(CT), and endoscopeic retrograde cholangiopancreatography(ERCP) were performed in all cases. CT was performed with contrast medium. The diagnosis of the disease was performed based on the results of the modalityes and laboratory data. The study population consisted of 35 patients with PCa(28 men, 8 women; range of age, 32 to 86 years), 18 with IPMN of pancreas(12 men, 6 women, rang of age, 54 to 79 years), and 25 with CP. PCa tumors were located in the head(18 cases), body and tail (19 cases) of the pancreass. 16 PCa tumor was histologyically confirmed at the time of operation or biopsy under endoscopic US. The remaining 21 pancreatic tumor, which were not histologyically examined, showed findings compatible with PCa on US, CT, endoscopeic retrograde cholangiopancreatography, and endoscopeic US, and these patients died of PCa with 3 to 30 months after diagnosis with a compatible clinical course. The stage of PCa was determined according to TNM classification of Pancreatic Carcinoma as set by the International Union Against Cancer. Thre was 2 patients with stageâ… , 1 patient with stageâ…¡, 3 patients with stageâ…¢, and 26 patients with stageâ…£tumors. Tumor size(TS) for PCa was determined according to the Classification of Pancreatic Carcinoma by the Japan Pancreas Socienty. The was 4 patients with TS1(less than 2cm), 15 patients with TS2(2 to 4 cm), and 15 patients with TS3(4 to 6 cm), and 3 patients with TS4 (more than 6 cm). The diagnosis of IPMN was made by various diagnostic modalityed or by operation according to the classification of the Armed Forces Institute of Pathology. The Patients with IPMN were divided into 2 groups, namely, a malignant and a benign group. The decision-making criteria of the malignant group of IPMN were required for at least 1 of 3 conditions under various modalities described below: (1) diameter of main pancreatic duct more than 7 mm; (2) size of the cystic lesionsmore than 30mm; (3) mural nodule more than 6 mm in size; and (4) borderline lesion or carcinoma that was histologically confirmed with operative materials. On the other hand, the benign group did not satisfy any condition described above or showed hyperplasia and adenoma that was histologically confirmed with surgical materials. Finally, 10 IPMN patients were judged as the malignant group, and 8 as the benign group. The diagnosis of CP was based on the clinical diagnostic criteria of the Japan Pancreas Society. 20 The 20 patients with CP were classified as definite based on the clinical diagnostic criteria and were followed up over a 12-month period with no clinical detection of the PCa tumor.â…¢Sample collection and DNA extractionApproxinmately 10ml samples of PPJ were collected by ERCP in 10ml centrifuge tubes and was stored at -80℃. Then, DNA was extracted from the supernatant with a phenol/chloroform mixture and precipitated with ethanol. The quantity of DNA was assessed from the absorbance at 260nm and the quality was evaluated from 260/280 absorbance ratio. The genomic DNA was stored at -80℃until use for genetic analysis. â…£MSP(â… ) Bisulfite treatmentDNA from supernatant of pancreatic juice was subjected to bisulfite treatment as described previously. Using a CpGenome DNA Modification Kit(Serologicals Corporation, USA), 1μg of pancreatic juice DNA was denatured with 2mol/L of NaOH at 37℃for 10 minutes, followed by incubation with 3mol/L of sodium bisulfite (pH 5.0) at 50℃for 16 hours. After that treatment, DNA was purified as recommended by the manufacturer, incubated with 3 mol/L of NaOH at room temperature for 5 minutes, precipitated with 10mol/L of ammonium acetate and. 90% ethanol, washed with 70% ethanol, and finally resuspended in 25μ1 of distilled water and stored at -80℃.(â…¡)MSPThe bisulfite-modified DNA was subject to MSP in a blinded manner using primer pairs specifically the methylated or unmethylated alleles for the respective genes as described recently Each PCR reaction mix consisted of a total volume of 25μl containing 10×PCR Gold Buffer(Applied Biosystems, USA), 10mM deoxynucleotide triphosphate mix(Applied Biosystems, USA), 3 to 4mM MgCl₂(Applied Biosystems, USA), 750nM each primer (Nihon Gene Research Laboratories Inc, Japan), 1.25 unit of AmipliTaq Gold DNA polymerase(Applied Biosystems, USA), and 2μl bisulfited DNA. The thermocycler conditions were in general as follows: 95℃for 10 mins, 35 cycles of 95℃for 1min, 61 to 64℃specific annealing temperature for 1min, 72℃for 1min, followed by a final extension at 72℃for 10 mins.(â…¢) ElectrophoresisThe MSP products were then subjected to horizontal gel electrophoresis through a 3% agarose gel, stained with SYBR Gold Nucleic Acid Gel Stain(Molecular Probes Inc, USA) and visualized with UV transillumination.(â…£)Realtime MSPWe then did realtime Q-MSP amplification of the promoter regions of NPTX2, CLDN5 and ppENK. MYOD1 served as internal reference. Only methylated analysis was performed. Q-MSP analysis was performed using a LightCycler (Roche Applied Science, Genmany) and the LightCycler-FastStart DNA Master SYBR Greenâ… Kit (Roche Applied Science, Genmany). Reactions were performed in a final volume of 10μ1, adjusted to 3 to 4 mM MgCl₂ and containing 500 nM of each primer (Nihon Gene Research Laboratories Inc, Japan) and 2μ1 of DNA template. PCR amplification was done by means of the following procedure: 95℃for 10 minutes, followed by 45 cycles at 95℃for 10 seconds, 61-64℃for 10 seconds, and 72℃for 10 seconds, followed by a final 10 minutes extension step at 72℃. The fluorescence signal of Q-MSP was generated by SYBR Greenâ… , a dye included in any double-stranded DNA during PCR amplification. As a positive controls, serial dilutions of cDNA obtained from MSP specific for each gene were used to construct a calibration curve. The calibration curve was used to set a plate specific threshold for positivity and to determine DNA equivalents for the results obtained. Multiple water blanks were included as negative controls. A subsequent dissociationcurve analysis checked the specificity of products. Only a sample with sufficient DNA input(ΔC＿T of MYOD1 is within 35cycles.) was accepted for a further statistical analysis. To determine the relative levels of methylation promoter DNA in each sample, we compared the values of each gene of interest with the values of the internal reference gene to obtain a ratio that was then multiplied by 10 for easier tabulation (target gene/internal reference gene×10) as described recently.â…¤Statistical analysis.Statistical evaluation was performed by using x² test and Fisher's exact test for enumeration data and independent t test for measurement data to assess relationship of methylation between pancreatic neoplasm. P＜0.05 was judged as significant.RESULTSâ… Detecting aberrant methylation of NPTX2, CLDN5 and ppENK genes by MSP in the pancreastic carcinorna cell linesPCR products of aberrant methylation in NPTX2, CLDN5 and ppENK genes were shown in all 9 PCa lines. PCR products of unmethylation in NPTX2 were shown in KLM1 and PK8 cell lines, in CLDN5 were shown in KLM1 and PK45H cell lines, and in ppENK were shown in KLM1, Panc1 and BxPC3 cell lines. The contrast group (DDW) was show neither methylation nor unmethylation. This result show that aberrant methylation in N-PTX2, CLDN5 and ppENK genes were widely existing in pancreastic carcinoma cell lines, and part of those cell lines shown uncomplete methylation.â…¡Detecting aberrant methylation of NPTX2, CLDN5 and ppENK genes by MSP in PPJBy MSP, in PPJ of 37 patients with pancreatic carcinoma, 23 cases in NPTX2, 27 cases in CLDN5, 18 cases in ppENK shown aberrant methylation. In PPJ of 25 patients with CP, 6 cases in NTPX2, 3 cases in CLDN5, 4 cases in ppENK, shown aberrant methylation. In 8 patients of the benign group of IPMN, 2 cases respectively in NPTX2, CLDN5 and ppENK genes shown aberrant methylation. In 10 patients of malignant group of IPMN, 7 cases in NPTX2, 7 cases in CLDN5, 5 cases in ppENK shown aberrant methylation.χ² test and Fisher's exact test revealed highly significant differences in NPTX2 between PCa and CP (P=0.003) , PCa and IPMN (P=0.023), the benign group and malignant group of IPMN (P=0.02).It shown highly significant differences in CLDN5 between PCa and CP (P=0.000), PCa and IPMN (P=0.002), IPMN and CP (P=0.042).It also shown highly significant differences in ppENK between PCa and CP (P=0.008).This revealed that detecting aberrant methylation in NPTX2, CLDN5, and ppENK by MSP shown high specificity and sensitivity, and might be used as a marker to help diagnose PCa.â…¢Detecting aberrant methylation of NPTX2, CLDN5 and ppENK genes by real time MSP in PPJLevel of aberrant methylation in NPTX2, CLDN5, and ppENK was 5.48±10.91,3.52±7.28, 1.61±2.36 (X10^-3) in PPJ of Patients with PCa, 19.07±32.73,2.50±4.02,1.58±2.14(X10^-3)in PPJ of malignant group of IPMN, 0.12±9.35,0.60±1.08,1.07±1.55 (X10^-3) in PPJ of benign group oflPMN, 0.86±1.66,0.26±0.58,0.63±1.03 (X10^-3) in PPJ of patients with CP. T test revealed highly significant differences in NPTX2 between PCa and CP (P=0.04), malignant group and benign group of IPMN (P=0.008). It shown highly significant differences in CLDN5 PCa and CP (P=0.03), malignant group and benign group of IPMN (P=0.00) . but it shown no significant differences in ppENK between PCa and CP. It revealed that detecting aberrant methylation in NPTX2 and CLDN5 might be used to distinguish benign and malignant diseases of pancreas. On the other hand, the level of aberrant methylation of 7 cases (18.9%) in NPTX2 and 9 cases (24.3%) in CLDN5 is obviously higher in PCa than in CP. It reveal that realtime MSP might be more valuable than MSP in diagnosing PCa.Further, we set a suitable cutoffof 1.0×10^-3,0.65×10^-3,0.7×10^-3 for NPTX2, CLDN5, and ppENK. By realtime MSP, in PPJ of patients with PCa, 20 cases in NPTX2, 20 cases in CLDN5, 20 cases in ppENK shown aberrant methylation. In PPJ of 25 patients with CP, 5 cases in NTPX2, 2 cases in CLDN5, 7 cases in ppENK, shown aberrant methylation. In 8 patients of the benign group of IPMN, 0 cases in NTPX2, 2 cases in CLDN5, 3 cases in ppENK,. In 10 patients of malignant group of IPMN, 4 cases in NPTX2, 6 cases in CLDN5, 6 cases in ppENK shown aberrant methylation.χ² test and Fisher's exact test revealed highly significant differences in NPTX2 between PCa and CP (P=0.007), PCa and IPMN (P=0.026). It shown highly significant differences between in CLDN5 PCa and CP (P=0.000), IPMN and CP (P=0.009). It also shown highly significant differences in ppENK between PCa and CP (P=0.042).â…£Relationship between aberrant methylation of NPTX2, CLDN5 andppENK genes in supernatant of PPJ samples and clinicopathologic parameters of PCaWe analyzed the relationship between aberrant methylation of NPTX2, CLDN5 and ppENK genes in PPJ samples and clinicopathologic parameters, including location, size and TNM stage of PCa. It shown significant difference only in CLDN5 between head, and body and tail of pancreastic tumor. It hinted that aberrant methylation in the three genes might be consider as having a pancreatic carcinoma. But using MSP we detected aberrant methylation in NPTX2, CLDN5, and ppENK genes in all of 2 cases at clinic stageâ… . It revealed that the three genes might to be useful as a marks to detect early pancreastic carcinoma. DISCUSSIONRecent studies have showed that epigenetic alterations including hypermethylation of tumor-related genes was involved in pancreatic cancer and played a important role in carcinogenesis. It is known that NPTX2 gene mediate the uptake of synaptic material and presynaptic toxins, CLDN5 gene is related to tight junction, and ppEKN gene is known to be a potent regulator of development, cell prolixferation and angiogenesis. It had been demonstrated that this three genes were methylated in over 90% of primary pancreatic cancer tissues and rarely methylated in normal pancreatic ductal epithelia. To evaluate the diagnostic significance of detecting aberrant methylation in PPJ from patients with PCa, IPMN, and CP, we investigated aberrant methylaiton in NPTX2 and CLDN5 genes.By MSP, PCR products of aberrant methylation in NPTX2, CLDN5 and ppENK genes were shown in all 9 PCa lines. PCR products of unmethylation in NPTX2 were shown in KLM1 and PK8 cell lines, in CLDN5 were shown in KLM1 and PK45H cell lines, and in ppENK were shown in KLM1, Pancl and BxPC3 cell lines. The contrast group (DDW) was show neither methylation nor unmethylation. This result show that aberrant methylation in NPTX2, CLDN5 and ppENK genes were widely existing in pancreastic carcinoma cell lines, and part of those cell lines shown uncomplete methylation. It revealed that aberrant methylation might general cases in process of PCa, and the level of aberrant methylation might be different in process.Recently, using MSP detecting aberrant methylation in PPJ might be highly specific as a marker for diagnosing PCa. Fukusima proved that 66.7%(30/45) of patients with PCa shown aberrant methylation in ppENK in PPJ. Sato proved that 67%(16/24), 46%(/1124), and 42%(10/24) of patients with PCa shown aberrant methylation in NPTX2, SARP2, and CLDN5 in PPJ, and detected no methylation in 13 patients with CP. Our study results is that by MSP, in PPJ of 37 patients with pancreatic carcinoma, 23 cases in NPTX2, 27 cases in CLDN5, 18 cases in ppENK shown aberrant methylation. In PPJ of 25 patients with CP, 6 cases in NTPX2, 3 cases in CLDN5, 4 cases in ppENK, shown aberrant methylation. In 8 patients of the benign group of IPMN, 2 cases respectively in NPTX2, CLDN5 and ppENK genes shown aberrant methylation. In 10 patients of malignant group of IPMN, 7 cases in NPTX2, 7 cases in CLDN5, 5 cases in ppENK shown aberrant methylation.χ² test and Fisher's exact test revealed highly significant differences in NPTX2 between PCa and CP (P=0.003), PCa and IPMN (P=0.023), the benign group and malignant group of IPMN (P=0.02). Itshown highly significant differences in CLDN5 between PCa and CP (P=0.000), PCa and IPMN (P=0.002), IPMN and CP (P=0.042). It also shown highly significant differences in ppENK between PCa and CP (P=0.008). This revealed that detecting aberrant methylation in NPTX2, CLDN5, and ppENK by MSP shown high specificity and sensitivity, and might be used as a marker to help diagnose PCa. From CP, IPMN to PCa, the rate of aberrant methylation in these three genes is gradually increased. Along with process of PCa, aberrant methylation in NPTX2 and CLDN5 significantly increased, this revealed that these three genes might be related to processs of PCa.Using MSP, it shown significant difference only in CLDN5 between head, and body and tail of pancreastic tumor. It hinted that sensitivity of detecting aberrant methylation in CLDN5 is related to location of pancreatic tumor. There was no significant difference between aberrant methylation in NPTX2 and ppENK genes and location, size, and TNM stage of PCa. It hinted that aberrant methylation might be a general cases in PCa, and be no related to clinicopathologic parameters of PCa. On the other hand, using MSP we detected aberrant methylation in NPTX2, CLDN5, and ppENK genes in all of 2 cases at clinic stageâ… . It revealed that the three genes is sensitive and specific enough to be used as a marks to detect early pancreastic carcinoma.Recent studies showed that quantification of methylation in cancer-related genes may be promising tools in diagnosis of neoplasm and reflect the progression of tumor. Yan L and et al demonstrated that high-level p 16(INK4a) promoter methylation was identified in most of pancreatic cancer but only in few of pancreatitis, and could used a marker to discriminate between patients with malignant and benign disease using Q-MSE Base on the results of MSP, we further investigated the relative quantification of hypermethylation in NPTX2 and CLDN5 genes in PPJ from patients with PCa, IPMNs, and CP using Q-MSP.T test revealed highly significant differences in NPTX2 between PCa and CP (P=0.04), malignant group and benign group of IPMN (P=0.008). It shown highly significant differences in CLDN5 PCa and CP (P=0.03), malignant group and benign group of IPMN (P=0.00). but it shown no significant differences in ppENK between PCa and CP. It revealed that detecting aberrant methylation in NPTX2 and CLDN5 might be used to distinguish benign and malignant diseases of pancreas. On the other hand, the level of aberrant methylation of 7 cases (18.9%) in NPTX2 and 9 cases (24.3 % ) in CLDN5 is obviously higher in PCa than in CP. It reveal that realtime MSP might be more valuable than MSP in diagnosing PCa. Along with process of PCa, level of aberrant methylation might be different in different phase. Realtime MSP might a better method to reveal actual contribution of aberrant methylation in process of PCa. On the other hand, the different level of aberrant methylation in these three genes may more actually reflect those genes taking part in process of PCa in different level. From CP, IPMN to PCa, it is shown that the number and level of aberrant methylation of those three genes is increased by realtime MSP. it is highly hinted that different level of aberrant methylation in NPTX2, CLDN5, and ppENK may contribute in PCa progress.Further, seting a suitable cutoff value, in PPJ of patients with PCa, 20 cases in NPTX2, 20 cases in CLDN5, 20 cases in ppENK shown aberrant methylation. In PPJ of 25 patients with CP, 5 cases in NTPX2, 2 cases in CLDN5, 7 cases in ppENK, shown aberrant methylation. In 8 patients of the benign group of IPMN, 0 cases in NTPX2, 2 cases in CLDN5, 3 cases in ppENK,. In 10 patients of malignant group of IPMN, 4 cases in NPTX2, 6 cases in CLDN5, 6 cases in ppENK shown aberrant methylation.χ² test and Fisher's exact test revealed highly significant differences in NPTX2 between PCa and CP (P=0.007), PCa and IPMN (P=0.026). It shown highly significant differences between in CLDN5 PCa and CP (P=0.000), IPMN and CP (P=0.009). It also shown highly significant differences in ppENK between PCa and CP (P=0.042). It revealed that detecting aberrant methylation by realtime MSP is suitable to be used as markers to diagnosing PCa, and realtime MSP might more veritablely revealed level of aberrant methylation in process of PCa.CONCLUSIONâ… By MSP, detecting aberrant methylation of NPTX2, CLDN5, and ppENK in PPJ was sensitive and specific enough to be used as marks to diagnose benign and malignant tumor of pancreas.â…¡By MSP, detecting aberrant methylation of NPTX2, CLDN5, and ppENK in PPJ may hint take part in process of PCa in different degree.â…¢By MSP, detecting aberrant methylation of NPTX2, CLDN5, and ppENK in PPJ might helpful to diagnose early pancreastic carcinoma.â…£By realtime MSP, detecting aberrant methylation of NPTX2, CLDN5, and ppENK in PPJ was sensitive and specific enough to be used as marks to diagnose benign and malignant tumor of pancreas.â…¤By realtime MSP, it is highly hinted that different level of aberrant methylation in NPTX2, CLDN5, and ppENK may contribute in PCa progress.â…¥By seting a suitable cutoff value, using realtime MSP, detecting aberrant methylation of NPTX2, CLDN5, and ppENK in PPJ is helpful to diagnose pancreatic disease, and more useful than MSR...