Diagnostic utility of aberrant methylation of tissue factor pathway inhibitor 2 in pure pancreatic juice for pancreatic carcinoma

Abstract

The tissue factor pathway inhibitor 2 (TFPI-2) is a Kunitz-type serine proteinase inhibitor. Recently, the aberrant methylation of TFPI-2 was detected frequently in pancreatic carcinoma (PCa) tissues but not in normal pancreatic tissues. We analyzed the aberrant methylation of TFPI-2 in the pure pancreatic juice (PPJ) aspirated endoscopically from patients with various pancreatic diseases. Using the highly sensitive methylation-specific polymerase chain reaction (MSP) and quantitative MSP (Q-MSP) assay, we investigated the aberrant methylation of TFPI-2 in nine human PCa cell lines and in the PPJ from patients with PCa, intraductal papillary mucinous neoplasms (IPMN) and chronic pancreatitis (CP). The incidence of aberrant TFPI-2 methylation was seven (77.8%) of nine PCa cell lines by Q-MSP. In cell lines, the expression of TFPI-2 mRNA by quantitative reverse transcription-polymerase chain reaction showed an inverse correlation to the aberrant methylation of TFPI-2. The incidence of aberrant TFPI-2 methylation in the PPJ was 21 (58.3%) of 36 PCa patients, three (17.6%) of 17 IPMN and one (4.8%) of 21 CP by MSP assay. Using a suitable cut-off value of 2.5 according to the receiver operating characteristic curve, the incidence of aberrant TFPI-2 methylation in the PPJ by real-time MSP was 18 (62.1%) of 29 PCa patients, one (5.1%) of 17 IPMN and three (14.3%) of 21 CP, respectively. The incidence of quantitative TFPI-2 hypermethylation in the PPJ with PCa was significantly higher than that with IPMN (P < 0.001) or CP (P < 0.001). Moreover, the aberrant methylation rate of TFPI-2 in the PPJ was 100%, as observed (6/6) in the PCa patients with liver metastasis, and 86.7% (26/30) in stages IVa + IVb of PCa by Q-MSP assay. These results suggest that promoter methylation of TFPI-2 in the PPJ may be a useful marker in the diagnosis and progression of PCa using an endoscopically feasible approach.

Figure 1

The amplification curves of tissue factor pathway inhibitor 2 (TFPI‐2) mRNA expression and TFPI‐2 aberrant methylation in the pancreatic cancer cell lines by quantitative analysis. (a) Serial 10‐fold dilutions of the BxPC‐3 cDNA gave the standard curves. The positive results of samples were judged according to the same melting temperatures and similar melting curves as the TFPI‐2 cDNAs. TFPI‐2 mRNA was expressed in KLM‐1, Mia‐PaCa‐2, PK‐8 and BxPC‐3 cell lines. (b) Serial 10‐fold dilutions of the PK‐9 methylation cDNA gave the standard curves. The positive results of samples were judged according to the same melting temperature and a similar melting curve as the methylated TFPI‐2 cDNAs. The aberrant methylation of TFPI‐2 was detected in KLM‐1, Mia‐PaCa‐2, PK‐45H, PK‐59, PK‐1, PK‐9 and BxPC‐3 cell lines. (c) The ratios of TFPI‐2:18S mRNA and hypermethylated TFPI‐2:MYOD1 in pancreatic cancer cell lines were compared. The expression of TFPI‐2 mRNA showed an inverse correlation to the aberrant methylation of TFPI‐2 by real‐time analysis.

Figure 2

Detection of the aberrant methylation of tissue factor pathway inhibitor 2 (TFPI‐2) in pure pancreatic juice (PPJ) from the patients with pancreatic adenocarcinoma (PCa), intraductal papillary mucinous neoplasm (IPMN) and chronic pancreatitis (CP), and primary PCa tissues by methylation‐specific polymerase chain reaction (MSP) analysis. (a) Using methylation and unmethylation specific primers, the 95‐bp methylated PCR product and 89‐bp unmethylated PCR product were detected in the PPJ from patients with PCa, IPMN and CP, and primary PCa tissues. (b) The aberrant methylation rate of TFPI‐2 in PCa, IPMN and CP was 58.3% (21/36), 17.6% (3/17) and 4.8% (1/21), respectively. The hypermethylation rate of TFPI‐2 was 14.3% (1/7) in benign IPMN and 20% (2/10) in malignant IPMN. The hypermethylation rate of TFPI‐2 in PCa was significantly higher than that in IPMN or CP, respectively (P < 0.01, P < 0.001).

Figure 3

Aberrant methylation of tissue factor pathway inhibitor 2 (TFPI‐2) in pure pancreatic juice (PPJ) from patients with pancreatic adenocarcinoma (PCa), intraductal papillary mucinous neoplasm (IPMN) and chronic pancreatitis (CP) by real‐time methylation‐specific polymerase chain reaction (MSP) analysis. (a) The ratio of hypermethylated TFPI‐2:MYOD1 in PCa, IPMN and CP was 6.62 ± 7.20, 0.58 ± 1.02 and 1.28 ± 1.73, respectively. The hypermethylation in PCa was significantly higher than that in IPMN (P < 0.01) or CP (P < 0.05). There was no significant difference between IPMN and CP. (b) The receiver operating characteristic curve of TFPI‐2 methylation in PCa diagnosis from the other pancreatic diseases. The suitable cut‐off value was selected as 2.5. FPR, false positive rate; TPR, true positive rate. (c) Using 2.5 as a suitable cut‐off value, the aberrant methylation rates of TFPI‐2 in PCa, IPMN and CP was 62.1% (18/29), 5.9% (1/17) and 14.3% (3/21), respectively. The incidence of hypermethylation for TFPI‐2 in PCa was significantly higher than that in IPMN (P < 0.001) or CP (P < 0.001).