Otic Bcl-2 and pro-apoptotic Bax in FU97 cells treated with As2O3. The mRNA and protein expression of Bcl-2 was downregulated in As2O3 treated cells (Fig. 4) but that of Bax was upregulated, which suggests that the effect of As2O3 in cell apoptosis was mediated by inhibition of constitutively activated STAT3 (Fig. 7).AFP(+) (n = 24) STAT3(+) STAT3(? (n = 11) (n = 13) p Age ,60 60 Sex Female Male Depth of MNS web invasion T1/T2 T3/T4 Pathology stage I I III V Lymph node metastasis No Yes 0(0 ) 11(58 ) 5(100 ) 8(42 ) 0.02* 3(27 ) 8(62 ) 8(72 ) 5(38 ) 0.09 3(25 ) 8(67 ) 9(75 ) 4(33 ) 0.04* 2(40 ) 9(47 ) 3(60 ) 10(53 1531364 ) 0.77 5(42 ) 6(50 ) 7(58 ) 6(50 ) 0.AFP(? (n = 24) STAT3(+) (n = 8) STAT3(? (n = 16) p3(38 ) 5(31 )5(62 ) 11(69 ) 0.Clinical Characteristics of the Selected PopulationThere were 34 male (70.8 ) and 14 female (29.2 ) patients, with a median age of 66 years(range, 45?3 years). The clinicopathological characteristics of the patients were summarized in Table 2. There were 48 patients had complete follow-up data, and the follow-up period was from 3 months to 60 months,with a mean period of 33.7 months. The overall survival time was defined as the months from the date of surgery to the date of death or loss follow-up.2(22 ) 6(40 )7(78 ) 9(60 ) 0.3(19 ) 5(63 )13(71 ) 3(37 ) 0.03*Immunohistochemical Expression of STATBecause we lack information on the expression of STAT3 in AFPGC, we determined its expression by immunohistochemical staining of AFPGC patient tissue. In the 24 AFPGC primary tumors, 11 were MedChemExpress Verubecestat positive (46 ) and 13 were negative (54 ) for STAT3 expression. In the 24 AFP-negative gastric cancer samples, 8 (33 ) primary tumors were positive and 16 (67 ) were negative for STAT3 expression. Moreover, despite the relatively low numbers of patients with complete data, STAT3 overexpression was significantly associated with the depth of invasion and lymph node metastasis (p,0.05) in the AFP-positive and -negative groups (Fig. 5, Table 2, Fig. 7).2(18 ) 6(46 )9(72 ) 7(54 ) 0.1(1 ) 7(54 )10(99 ) 6(46 ) 0.02*Figures in parentheses are percentages. *Considered to be statistically significant. doi:10.1371/journal.pone.0054774.tNovel Therapy for AFP-Producing Gastric CancersFigure 5. Representative immunohistochemical staining in serial sections of poorly differentiated adenocarcinoma of the stomach from patients positive for AFP (magnification 6100). (A) Immunostaining for AFP. (B) Strong STAT3 immunostaining with brown granular deposits in the cytoplasm and nuclei. (C) Negative control immunohistochemical staining for AFP. (D) Negative control immunohistochemical staining for STAT3. doi:10.1371/journal.pone.0054774.gExpression of AFP and STAT3 Associated with Poor Prognosis of Gastric CancerThe median survival time of AFP-positive patients was 23 months (95 confidence interval, 16?0 months). which was significantly shorter than that in the AFP-negative patients, 53 months (95 1317923 confidence interval, 47?9 months) (P,0.05).The median survival time in the STAT3-positive group was 38 months (95 confidence interval, 29?7 months), which was significantly shorter than that in the STAT3-negative group, 54 months (95 confidence interval, 47?1 months) (P,0.05, Fig. 6A and B, Fig. 7).Furthermore, survival was lower for AFP and STAT3 double-positive patients than with expression of AFP or STAT3 alone (P,0.05, Fig. 6C and D, Fig. 7). In patients with AFP and STAT3 double-positive expression the median survival time was 22 months (95 confidence interval.Otic Bcl-2 and pro-apoptotic Bax in FU97 cells treated with As2O3. The mRNA and protein expression of Bcl-2 was downregulated in As2O3 treated cells (Fig. 4) but that of Bax was upregulated, which suggests that the effect of As2O3 in cell apoptosis was mediated by inhibition of constitutively activated STAT3 (Fig. 7).AFP(+) (n = 24) STAT3(+) STAT3(? (n = 11) (n = 13) p Age ,60 60 Sex Female Male Depth of invasion T1/T2 T3/T4 Pathology stage I I III V Lymph node metastasis No Yes 0(0 ) 11(58 ) 5(100 ) 8(42 ) 0.02* 3(27 ) 8(62 ) 8(72 ) 5(38 ) 0.09 3(25 ) 8(67 ) 9(75 ) 4(33 ) 0.04* 2(40 ) 9(47 ) 3(60 ) 10(53 1531364 ) 0.77 5(42 ) 6(50 ) 7(58 ) 6(50 ) 0.AFP(? (n = 24) STAT3(+) (n = 8) STAT3(? (n = 16) p3(38 ) 5(31 )5(62 ) 11(69 ) 0.Clinical Characteristics of the Selected PopulationThere were 34 male (70.8 ) and 14 female (29.2 ) patients, with a median age of 66 years(range, 45?3 years). The clinicopathological characteristics of the patients were summarized in Table 2. There were 48 patients had complete follow-up data, and the follow-up period was from 3 months to 60 months,with a mean period of 33.7 months. The overall survival time was defined as the months from the date of surgery to the date of death or loss follow-up.2(22 ) 6(40 )7(78 ) 9(60 ) 0.3(19 ) 5(63 )13(71 ) 3(37 ) 0.03*Immunohistochemical Expression of STATBecause we lack information on the expression of STAT3 in AFPGC, we determined its expression by immunohistochemical staining of AFPGC patient tissue. In the 24 AFPGC primary tumors, 11 were positive (46 ) and 13 were negative (54 ) for STAT3 expression. In the 24 AFP-negative gastric cancer samples, 8 (33 ) primary tumors were positive and 16 (67 ) were negative for STAT3 expression. Moreover, despite the relatively low numbers of patients with complete data, STAT3 overexpression was significantly associated with the depth of invasion and lymph node metastasis (p,0.05) in the AFP-positive and -negative groups (Fig. 5, Table 2, Fig. 7).2(18 ) 6(46 )9(72 ) 7(54 ) 0.1(1 ) 7(54 )10(99 ) 6(46 ) 0.02*Figures in parentheses are percentages. *Considered to be statistically significant. doi:10.1371/journal.pone.0054774.tNovel Therapy for AFP-Producing Gastric CancersFigure 5. Representative immunohistochemical staining in serial sections of poorly differentiated adenocarcinoma of the stomach from patients positive for AFP (magnification 6100). (A) Immunostaining for AFP. (B) Strong STAT3 immunostaining with brown granular deposits in the cytoplasm and nuclei. (C) Negative control immunohistochemical staining for AFP. (D) Negative control immunohistochemical staining for STAT3. doi:10.1371/journal.pone.0054774.gExpression of AFP and STAT3 Associated with Poor Prognosis of Gastric CancerThe median survival time of AFP-positive patients was 23 months (95 confidence interval, 16?0 months). which was significantly shorter than that in the AFP-negative patients, 53 months (95 1317923 confidence interval, 47?9 months) (P,0.05).The median survival time in the STAT3-positive group was 38 months (95 confidence interval, 29?7 months), which was significantly shorter than that in the STAT3-negative group, 54 months (95 confidence interval, 47?1 months) (P,0.05, Fig. 6A and B, Fig. 7).Furthermore, survival was lower for AFP and STAT3 double-positive patients than with expression of AFP or STAT3 alone (P,0.05, Fig. 6C and D, Fig. 7). In patients with AFP and STAT3 double-positive expression the median survival time was 22 months (95 confidence interval.