Ed mucin) is related with invasive proliferation of the tumors and poor outcome of the patients, whereas the expression of the MUC2 mucin (intestinal type secretory mucin) is related with the non-invasive proliferation of the tumors and a favorable outcome for the patients [4,5]. Our previous study showed thatMUC4 and MUC1 Expression in Early Gastric CancersFigure 1. The difference in antibody specificity between anti-human MUC4 monoclonal antibodies (MAbs), 8G7 and 1G8. A: MUC4 mRNA was detected in the two gastric cancer cell lines, SNU-16 and NCI-N87. PANC1 and CAPAN1 cells were used as a negative and positive control, respectively. B: Cell lysates of SNU-16 and NCI-N87 were immunoblotted and detected by the indicated antibodies, respectively. A-tubulin served as a loading control. C: Formalin-fixed SNU-16 and NCI-N87 cells were processed for immunocytochemistry using the MAbs, 8G7 and 1G8, respectively. Original magnification 6400. doi:10.1371/journal.pone.0049251.gMUC1 expression in gastric cancers is a poor prognostic factor [6]. MUC4 was first reported as tracheobronchial mucin [7] and is a membrane-associated mucin [8]. In our study series, the expression of MUC4 in intrahepatic cholangiocarcinoma, pancreatic ductal adenocarcinoma, extrahepatic bile duct carcinoma, lung adenocarcinoma, and oral squamous cell carcinoma was an independent factor for poor prognosis and is a useful marker to predict the outcome of the patients [5,9,10,11,12,13]. Unfortunatly, there are few studies of the MUC4 expression profile in human gastric cancer. In the present study, we examined the expression profiles of MUC4 as well as MUC1 in early gastric cancer tissues, and found that MUC4 and MUC1 expression in the early gastric cancers would become poor prognostic factors 18055761 by lymph vessel invasion, blood vessel invasion and lymph node metastasis. As anti-MUC4 monoclonal antibodies (MAbs), 8G7 and 1G8, are known to detect different sites of MUC4 molecule. The MAb 8G7 recognizes a tandem repeat sequence (STGDTTPLPVTDTSSV) of the human MUC4a subunit [14]. The MAb 
1G8 is raised PS-1145 biological activity against the rat sequence (rat ASGP-2), and recognizes an epitope on the rat ASGP-2 subunit, which corresponds to the human MUC4b subunit, and shows a cross reactivity with human samples [15]. Thus, a special attention was paid to the comparison of two anti-MUC4 MAbs by Western blotting and IHC of two gastric cancer cell lines, before the IHC study of human gastric cancer tissues. Moreover, since there is controversy regarding the prognostic significance of these anti-MUC4 MAbs, a literature review of MUC4 expression in various cancers was also performed.Materials and Methods Patients and Tissue SamplesGastrectomy specimens of 104 early gastric cancers, which show submucosal invasion, pT1b2, with or without lymph node metastasis, were retrieved from the file between 1994 and 2008 of the Kagoshima-shi Medical Association Hospital. The mean age of the patients was 65.7 (S.D., 9.8; range, 39?2 years; median age, 66 years); 64 cases were male, and 40 cases were female. This Study was conducted in accordance with the guiding principles of the Declaration of Helsinki, and approved by the Ethics Committee for Kagoshima-shi Medical Association Hospital (KMAH 2011-02-02). Informed, TA-02 price written consent was obtained from all patients. In the cases with more than two histological types mixed in one lesion, each histological pattern was evaluated independently, according to the Japanese Classification.Ed mucin) is related with invasive proliferation of the tumors and poor outcome of the patients, whereas the expression of the MUC2 mucin (intestinal type secretory mucin) is related with the non-invasive proliferation of the tumors and a favorable outcome for the patients [4,5]. Our previous study showed thatMUC4 and MUC1 Expression in Early Gastric CancersFigure 1. The difference in antibody specificity between anti-human MUC4 monoclonal antibodies (MAbs), 8G7 and 1G8. A: MUC4 mRNA was detected in the two gastric cancer cell lines, SNU-16 and NCI-N87. PANC1 and CAPAN1 cells were used as a negative and positive control, respectively. B: Cell lysates of SNU-16 and NCI-N87 were immunoblotted and detected by the indicated antibodies, respectively. A-tubulin served as a loading control. C: Formalin-fixed SNU-16 and NCI-N87 cells were processed for immunocytochemistry using the MAbs, 8G7 and 1G8, respectively. Original magnification 6400. doi:10.1371/journal.pone.0049251.gMUC1 expression in gastric cancers is a poor prognostic factor [6]. MUC4 was first reported as tracheobronchial mucin [7] and is a membrane-associated mucin [8]. In our study series, the expression of MUC4 in intrahepatic cholangiocarcinoma, pancreatic ductal adenocarcinoma, extrahepatic bile duct carcinoma, lung adenocarcinoma, and oral squamous cell carcinoma was an independent factor for poor prognosis and is a useful marker to predict the outcome of the patients [5,9,10,11,12,13]. Unfortunatly, there are few studies of the MUC4 expression profile in human gastric cancer. In the present study, we examined the expression profiles of MUC4 as well as MUC1 in early gastric cancer tissues, and found that MUC4 and MUC1 expression in the early gastric cancers would become poor prognostic factors 18055761 by lymph vessel invasion, blood vessel invasion and lymph node metastasis. As anti-MUC4 monoclonal antibodies (MAbs), 8G7 and 1G8, are known to detect different sites of MUC4 molecule. The MAb 8G7 recognizes a tandem repeat sequence (STGDTTPLPVTDTSSV) of the human MUC4a subunit [14]. The MAb 1G8 is raised against the rat sequence (rat ASGP-2), and recognizes an epitope on the rat ASGP-2 subunit, which corresponds to the human MUC4b subunit, and shows a cross reactivity with human samples [15]. Thus, a special attention was paid to the comparison of two anti-MUC4 MAbs by Western blotting and IHC of two gastric cancer cell lines, before the IHC study of human gastric cancer tissues. Moreover, since there is controversy regarding the prognostic significance of these anti-MUC4 MAbs, a literature review of MUC4 expression in various cancers was also performed.Materials and Methods Patients and Tissue SamplesGastrectomy specimens of 104 early gastric cancers, which show submucosal invasion, pT1b2, with or without lymph node metastasis, were retrieved from the file between 1994 and 2008 of the Kagoshima-shi Medical Association Hospital. The mean age of the patients was 65.7 (S.D., 9.8; range, 39?2 years; median age, 66 years); 64 cases were 
male, and 40 cases were female. This Study was conducted in accordance with the guiding principles of the Declaration of Helsinki, and approved by the Ethics Committee for Kagoshima-shi Medical Association Hospital (KMAH 2011-02-02). Informed, written consent was obtained from all patients. In the cases with more than two histological types mixed in one lesion, each histological pattern was evaluated independently, according to the Japanese Classification.
