Clinical utility of quantitative RT-PCR targeted to alpha1,4-N-acetylglucosaminyltransferase mRNA for detection of pancreatic cancer

Abstract

alpha1,4-N-Acetylglucosaminyltransferase (alpha4GnT) is a glycosyltransferase responsible for the biosynthesis of alpha1,4-GlcNAc-capped O-glycans, and is frequently expressed in pancreatic cancer cells but not peripheral blood cells. In the present study, we tested the clinical utility of alpha4GnT mRNA expressed in the mononuclear cell fraction of peripheral blood as a biomarker of pancreatic cancer. Total RNA isolated from the peripheral blood mononuclear cells from 55 pancreatic cancer patients, 10 chronic pancreatitis patients, and 70 cancer-free volunteers was analyzed quantitatively by reverse transcription-polymerase chain reaction with primers specific for alpha4GnT, and the expression level of alpha4GnT mRNA relative to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was measured. When the ratio of alpha4GnT to GAPDH transcripts exceeded a defined cut-off value, patients were considered to have pancreatic cancer. By these standards, 76.4% of the pancreatic cancer patients were detected by this assay. A strong correlation was obtained between positivity in this assay and the expression of alpha4GnT protein detected immunohistochemically in pancreatic cancer tissues resected subsequently, suggesting that alpha4GnT mRNA detected in the peripheral blood is derived from circulating pancreatic cancer cells. Although increased levels of alpha4GnT mRNA was detected in 40.0% of chronic pancreatitis patients and 17.1% of cancer-free volunteers, the expression levels were significantly lower than those seen in pancreatic cancer patients. These results suggest that quantitative analysis of alpha4GnT mRNA expressed in the mononuclear cell fraction of peripheral blood will contribute to the detection of pancreatic cancer.

Figure 1

Expression of α4GnT protein in the normal and neoplastic pancreatic tissues. α4GnT was detected by immunohistochemistry using the antiα4GnT antibody I17K. α4GnT is not expressed in the normal pancreatic duct (a), whereas it is expressed in the Golgi region of pancreatic ducts exhibiting PanIN‐IB (b). The α4GnT protein is also expressed in the pancreatic ducts with PanIN‐II found in chronic pancreatitis (c). In the pancreatic carcinoma, α4GnT protein is detected in well differentiated (d), moderately differentiated (e), and poorly differentiated (f) adenocarcinomas. Scale bar = 100 µM.

Figure 2

Quantitative RT‐PCR assay targeting α4GnT mRNA. (a) A standard curve for α4GnT was constructed by plotting serially diluted α4GnT cDNA, pcDNAI‐α4GnT (black dots), where unknown samples from patients or cancer‐free volunteers are indicated as red dots. (b) ROC curve was created by plotting the expression level of α4GnT mRNA in the peripheral blood from 55 pancreatic cancer patients and 70 cancer‐free volunteers. Arrow denotes the cutoff value of 10.5, which best discriminates pancreatic cancer patients from cancer‐free volunteers with 76.4% sensitivity and 82.9% specificity.

Figure 3

Scatter plots indicating the expression level of α4GnT mRNA in the mononuclear cell fraction of peripheral blood measured by the quantitative RT‐PCR. Small and large circles represent one and 10 individuals, respectively. Horizontal bars indicate mean ± SE. *P < 0.05, **P < 0.001.