Introduction 1 16 20 3 4 23 Saccharomyces cerevisiae 10 17 18 24 27 28 4 25 17 7 20 5 22 19 9 32 33 12 13 31 Materials and methods Subjects n n n Pathology 21 13 31 Tissue microarray Representative areas of solid tumours were identified in HE-stained sections of the selected tumour cases and a 2-mm-in-diametre tissue core per donor block was punched out and transferred to a recipient block with a maximum of 48 cores using a Tissue Microarrayer (AZUMAYA KIN-1, Japan). Four-micrometre-thick sections were consecutively incised from the recipient block and transferred to polylysine-coated glass slides. HE staining was performed on TMA for confirmation of tumour tissue. Cell lines and culture 2 Immunohistochemistry 11 One hundred cells were randomly selected and counted from five representative fields of each section blindly by three independent observers (Takano Y, Li XH and Zheng HC). The positive percentage of counted cells was graded semi-quantitatively according to a four-tier scoring system: negative (−), 0~5%; weakly positive (+), 6~25%; moderately positive (++), 26~50%; and strongly positive (+++), 51~100%. Western blot Fifty-microgramme denatured protein was separated on an SDS-polyacrylamide gel (10% acrylamide) and transferred to Hybond membrane (Amersham, Germany), which was then blocked overnight in 5% milk in TBST. For immunoblotting, the membrane was incubated for 1 h with mouse anti-parafibromin antibody as described above. Then, it was rinsed by TBST and incubated with anti-mouse immunoglobulin G conjugated to horseradish peroxidase (DAKO, CA, USA, 1:1,000) for 1 h. Bands were visualised with X-ray film (Fujifilm, Japan) by ECL-Plus detection reagents (Amersham, Germany). After that, membrane was washed with WB Stripping Solution (pH 2–3, Nacalai, Tokyo, Japan) for 30 min and treated as described above except mouse anti-β-actin antibody (Sigma, MO, USA, 1:5,000) as an internal control. Statistical analysis Spearman Kaplan Meier P Results Parafibromin expression in gastric tumours and carcinoma cell lines 1 2 p 1 2 p p p p Fig. 1 a a b c d e b Lane #1 #2 #3 #4 #5  Fig. 2 a c b a c d  Table 1 Parafibromin expression in gastric tissue samples Groups Number Parafibromin expression − + ++ +++ PR (%) Gastritis 49 0 1 7 41 a Gastric adenoma 45 9 5 8 23 b Gastric carcinoma 508 275 55 60 118 45.9 PR a p b p Table 2  Relationship between parafibromin expression and clinicopathological features of gastric carcinomas Clinicopathological features Number Parafibromin expression − + ++ +++ PR (%) Rs p Age (years)   <65 209 125 24 24 36 40.2 0.095 <0.05   ≥65 299 150 31 36 82 49.8 Sex   Male 354 188 37 38 91 46.9 0.054 >0.05   Female 154 87 18 22 27 43.5 Tumour size (cm)   <4 263 116 26 34 85 55.9 −0.237 <0.001   ≥4 245 159 27 26 33 35.1 Depth of invasion is−1 263 102 30 40 91 61.2 −0.344 <0.001 2-4 245 173 25 20 27 29.4 Lymphatic invasion   − 331 157 42 45 87 52.6 −0.168 <0.001   + 177 118 13 15 31 33.3 Venous invasion   − 443 236 49 51 107 46.7 −0.051 >0.05   + 65 39 6 9 11 40.0 Lymph node metastasis   − 317 138 38 44 97 56.5 −0.285 <0.001   + 191 137 17 16 21 28.3 UICC staging   0–I 292 123 36 41 92 57.9 −0.292 <0.001   II-IV 216 152 19 19 26 29.6 Lauren’s classification   Intestinal-type 273 108 34 38 93 60.4 −0.322 <0.001   Diffuse-type 225 157 21 22 25 30.2 PR T is T 1 T 2 T 3 T 4 UICC Univariate and multivariate survival analysis 3 Kaplan Meier p 3 3 p p 3 Fig. 3 Kaplan Meier a b EGC c AGC  Table 3 Multivariate analysis of clinicopathological variables for survival with gastric carcinomas Number Clinicopathological parametres Relative risk (95%CI) p A Age (≥65 years) 1.929 (1.357–2.743) <0.001 B Sex (female) 1.463 (0.983–2.179) >0.05 C Tumour size (≥4 cm) 1.606 (1.013–2.547) <0.05 D 2–4 6.530 (3.110–13.710) <0.001 E Lymphatic invasion (+) 2.626 (1.516–3.374) <0.001 F Venous invasion (+) 0.959 (0.633–1.452) >0.05 G Lymph node metastasis (+) 2.773 (1.525–5.043) <0.01 H UICC staging (II-IV) 0.294 (0.139–0.622) <0.01 I Lauren’s classification (diffuse-type) 1.796 (1.212–2.661) <0.01 J Parafibromin expression (+~+++) 0.792 (0.529–1.187) >0.05 CI UICC Discussion 4 5 8 17 19 17 2 5 6 8 14 19 17 2 6 10 17 34 29 30 20 25 26 19 15 28 Until now, there is yet no paper describing the prognostic significance of parafibromin expression in malignancies. Here, for the first time, we analysed the relation of parafibromin expression with the survival rate of 508 patients with gastric carcinoma. The results revealed a close link between its loss and worse survival. If stratified according to the depth of invasion, the significant link disappeared, indicating that the relationship between parafibromin expression and prognosis depends on the depth of invasion. The multivariate analysis demonstrated that age, depth of invasion, lymphatic invasion, lymph node metastasis, UICC staging and Lauren’s classification but not parafibromin expression, venous invasion or sex were independent prognostic factors for carcinomas. These findings suggested that parafibromin expression is a promising indicator for the favorable prognosis of gastric carcinoma patients, albeit not independent. 33 5 19 In summary, downregulated parafibromin expression might play an important role in malignant transformation of gastric epithelial cells. Its reduced expression was closely related to growth, invasion, metastasis and worse prognosis of gastric carcinomas. Its expression could be employed to differentiate the intestinal- and diffuse-type carcinomas and underlay the molecular mechanism about the differentiation of both carcinomas. It was considered as a promising marker to indicate the pathobiological behaviours and prognosis of gastric carcinomas.