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It is difficult to distinguish pancreatic ductal adenocarcinoma (PDAC) from chronic pancreatitis (CP) when stricture is present in the pancreatic duct. Endoscopic brushing cytology is a convenient method for investigating strictures in the pancreatic duct, however, the diagnostic sensitivity of this method for PDAC is reported to be low (40–70%). Recently, we revealed that MSX2 is frequently expressed in PDAC cells but not in normal cultured pancreatic duct or stellate cells. Thus, we analyzed MSX2 expression levels in brushing samples to examine whether this would differentiate PDAC from CP. Cytologic brushing specimens were obtained from pancreatic duct strictures during endoscopic retrograde cholangiopancreaticography in 82 patients. The brushing fluid was subjected to cytological diagnosis and RNA extraction. The expression level of MSX2 was evaluated by one-step real-time RT-PCR. MSX2 expression levels were significantly higher in PDAC than in CP (P = 0.0000007), and the expression level was associated with positive cytology (P = 0.013). The sensitivity, specificity, and diagnostic accuracy for PDAC of cytology and MSX2 expression in ductal strictures were: 47.4%, 100%, and 63.4%, and 73.7%, 84.0%, and 79.3%, respectively. The sensitivity and accuracy of MSX2 expression levels for diagnosis were much higher than those of cytology. This suggests that the evaluation of MSX2 levels in endoscopic retrograde cholangiopancreaticography brushing samples would be useful for distinguishing PDAC from CP. (Cancer Sci 2011; 102: 157–161)
Pancreatic ductal adenocarcinoma is one of the most malignant gastrointestinal tumors. Once pancreatic cancer is clinically evident, it progresses rapidly to develop metastatic lesions, frequently by the time of diagnosis. Furthermore, these tumors are usually resistant to conventional chemotherapy and radiation therapy. Therefore, accurate methods for detection of early PDAC are required to improve its poor prognosis. The discrimination of early pancreatic carcinoma from CP is sometimes very difficult because both diseases show similar stricture in the pancreatic duct. Thus, differential diagnosis for PDAC and CP is of critical importance to avoid delaying treatment of early pancreatic cancer. Endoscopic ultrasound-guided fine needle aspiration biopsy is an effective technique in the diagnosis and staging PDAC, and accuracy for the diagnosis of malignancy is high with sensitivity ranging from approximately 80% to 90%.(1–3) However, the availability of this procedure is restricted because the necessary equipment is expensive and the technique itself is difficult. Endoscopic pancreatic duct brushing is a convenient method for strictures in the main pancreatic duct or in the second branch. However, the diagnostic sensitivity of this method for PDAC is reported to be low (40–70%)(4–7) compared with that of EUS-FNAB.
A member of the homeobox genes (Hox genes) family, MSX2 is present in a variety of sites, including premigratory cranial neural crest, tooth, retina and lens, apical ectodermal ridge, and mammary gland.(8–11) In the development of these organs, the expression patterns of this gene suggest its active involvement in epithelial–mesenchymal interactions.(12,13) Enhanced levels of transcripts for MSX2 have been shown in a variety of carcinoma cell lines of epithelial origin compared to their corresponding normal tissues.(14) Recently, we have shown that MSX2 was frequently expressed in pancreatic carcinoma cell lines and PDAC tissues but not in benign cultured cells, including PSC, which are largely responsible for fibrogenesis of CP(15) or normal human pancreatic tissues,(16,17) suggesting that detection of MSX2 could be a useful marker for differentiating PDAC from CP. Therefore, we investigated the mRNA expression level of MSX2 in 82 endoscopic brushing samples from stricture of the pancreatic duct to examine whether MSX2 expression would distinguish malignant from benign pancreatic disease and could improve the diagnostic yield of brush cytology.
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- Materials and Methods
The differential diagnosis between PDAC and CP is generally difficult because both appear to be solid masses, like lesions with fibrotic changes, with stricture in the pancreatic duct. Although EUS-FNAB is an effective technique in the diagnosis of pancreatic mass, lesions presenting as strictures without a demonstrable mass should be investigated by other methods such as cytologic examination of pancreatic juice or pancreatic duct brushing. In addition, expensive equipment and difficulties with the technique restrict its availability. However, brushing cytology is a convenient method for pancreatic ductal stricture,(7) although its sensitivity is lower than that of EUS-FNAB.(1–7) To improve the sensitivity of brushing cytology for PDAC, we investigated MSX2 expression in brushing samples. In our previous study, MSX2 was expressed in PDAC cells but not in normal pancreatic duct cells or normal cultured cells, including PSC,(16,17) suggesting that the detection of MSX2 expression would be useful to discriminate PDAC from CP. As expected, we clearly revealed in the current study that: (i) expression of MSX2 mRNA was significantly higher in microdissected PDAC cells than non-cancerous cells; (ii) significantly high expression levels of MSX2 mRNA were detected in brushing specimens from PDAC compared to those from CP; and (iii) diagnostic sensitivity and accuracy by analyzing MSX2 expression levels were much higher than those by cytological examination.
Many genetic alterations have been shown in PDAC, the most common genetic alterations of PDAC include oncogene (K-ras), and tumor suppressor gene mutations (p16, p53, SMAD4).(19) Of these, K-ras mutation occurs at very high frequency.(20) However, K-ras muations are detected in CP as well as PDACs,(21) therefore, it is difficult to use the K-ras mutation as a tool to differentiate PDAC from CP. For example, K-ras mutation was analyzed in brush samples with 34 cases of PDAC and 11 with CP and was frequently detected in both cancer (87%) and pancreatitis (40%).(4) However, Parsi et al. measured DNA concentrations of methylated cyclin D2, NPTX2, and TFPI2 promoter in biliary or pancreatic duct brush samples to improve the diagnostic accuracy of brushing cytology. They revealed that nine of 66 (13.6%) disease controls, as well as 30 of 41 (73.2%) patients with PDAC, had methylation of one or more genes in their endoscopic brush samples.(22) This sensitivity or false positive rate for diagnosis of PDAC is similar to our current results. In addition, the diagnostic sensitivity or accuracy by our evaluation of MSX2 expression compares favorably to other markers such as telomerase(23) or MUC1(24) that have been reported to differentiate benign from malignant pancreatic diseases. In this context, the measurement of MSX2 expression levels in the brushing samples would be a good tool to help distinguish PDAC from benign pancreatic duct, in addition to other molecular analyses as above.
MSX2 is suggested to be a downstream target for the ras signaling pathway(25) and is expressed in carcinoma of epithelial origin,(14) suggesting the association of this gene with the development of pancreatic carcinoma, as pancreatic carcinoma harbors frequent mutation of the K-ras gene.(19,20) Accordingly, we have shown that PDAC tissues frequently expressed MSX2 protein and this correlated with less differentiation of carcinoma cells and venous infiltration. Also, forced expression of MSX2 in pancreatic carcinoma cells enhanced the aggressive phenotype through epithelial to mesenchymal transition.(26) This evidence suggested that MSX2 is particularly expressed in invading carcinoma cells. Thus, a high level of MSX2 expression was likely to be frequently detected in ductal strictures that were caused by invading PDAC cells.
In the current study, only four of 25 cases (16%) of CP showed positive MSX2 expression. Chronic pancreatitis is generally known as a risk factor for PDAC and contains premalignant ductal changes, such as PanIN, that show K-ras mutation or other molecular alterations.(27) Thus, molecular markers might tend to show relatively high false positive rates in CP.(4,22) This is likely to be favorable to predict the occurrence of PDAC in CP, but not to avoid unnecessary procedures. In contrast, MSX2 mRNA expression was shown to be below the detection level in premalignant lesions of the pancreas such as adenoma or hyperplastic lesions of intraductal papillary mucinous neoplasm.(17) This supports the fact that MSX2 analysis resulted in low false positive rates in CP. Therefore, repeated evaluation of MSX2 levels in CP patients might help in the early detection for occurrence of PDAC with high specificity, as MSX2 is only expressed in carcinoma cells. Alternatively, CP patients with positive MSX2 expression might have small malignant lesions that could not be detected by standard imaging. Thus, in such cases, careful follow-up by imaging examination would be necessary.
Cytology is the gold standard to diagnose PDAC preoperatively. However, inadequate sampling or a low rate of neoplastic cells in the specimens would prevent accurate diagnosis.(7) Thus, additional methods such as molecular marker analysis might help to encourage efforts to repeat sampling to obtain a cytological diagnosis, or to select EUS-FNAB if a mass is present. As shown in Figure 4, the validation of MSX2 expression levels was useful to diagnose PDAC when cytology alone could not confirm the presence of cancer cells, indicating that MSX2 analysis itself can be a good molecular marker and assist cytological examination. However, this validation could not provide positive results in PDAC located in the tail, and neither could cytological examination, suggesting the technical difficulty in sampling from the pancreatic tail. In addition, 13 of 95 cases (13.7%) failed to gain the brushing specimen for cytological examination and MSX2 analysis because of severe ductal stricture. To increase the sensitivity or diagnostic accuracy for PDAC by MSX2 analysis, further examination for ductal stricture in the tail and improvement of equipment for endoscopic brushing would be required.
There were three PDAC cases that showed positive cytology but negative MSX2 expression. The reason why the presence of carcinoma cells in the brushing specimens did not provide positive MSX2 expression levels is not clear. However, the lack of carcinoma cells in the RNA extraction buffer might partly explain this discrepancy, because all of the carcinoma cells in the brushing specimens were placed in saline solution for cytology. Alternatively, these PDAC cells might express very low levels of MSX2 mRNA. The combination of MSX2 analysis with cytology or clinical diagnosis by imaging examination would be important to avoid false negative diagnoses.
In conclusion, MSX2 was significantly expressed in brush samples of PDAC compared to those of CP. The sensitivity for malignancy and accuracy for diagnosis were much higher than cytology, suggesting that the evaluation of MSX2 expression levels is a good technique for differentiating PDAC from CP when stricture is present in the pancreatic duct.