Pre-treatment serum samples were obtained from 291 patients with histologically proven gastric adenocarcinoma and from 73 healthy individuals. Double cancer was excluded. All patients with gastric cancer were surgically treated (between July 2011 and July 2013) at the Toho University Omori Hospital (n=76) and the Chiba Cancer Center (n=215). Among these, 184 were diagnosed with stage I, 28 with stage II, 29 with stage III, and 50 with stage IV gastric cancer. The patients included 201 men and 90 women (mean age, 67.5 years; range, 36-93 years). Written informed consent was obtained from all patients. The samples were anonymized. Each serum sample was centrifuged at 3,000 x g, at room temperature for 5 min, and the resulting supernatant was stored at -80˚C until further analysis. Due care was taken to avoid the repeated thawing and freezing of samples. The present study was approved by the institutional review boards at the Chiba Cancer Center (approval no. #21-26) and the Toho University School of Medicine (approval nos. #22-112 and #22-047).

RalA construct inserted in pET28 plasmid and expressing the N-terminal His-tagged protein was provided by Dr Jian-Ying Zhang (The University of Texas, El Paso, TX). The details of this procedure have been described previously (9). Sera from patients and healthy controls were analyzed by the previously established ELISA (9). Briefly, purified recombinant proteins were placed in 96-well microtiter plates (Nunc MaxiSorp; Thermo Fisher Scientific, Inc.). RalA was diluted in phosphate-buffered saline (PBS) to a final concentration of 1.0 µg/ml and added to the plates (100 µl/well), which were then incubated overnight at 4˚C. PBS was used as a control. After two washes with PBS, proteins were blocked using 200 µl of PBS, containing 1% bovine serum albumin and 5% sucrose, at room temperature for 3 h. All human sera were diluted (1:100) in PBS containing 0.15% Tween-20, 1% casein, and 0.2 mg/ml E. coli extract. Then, 100 µl diluted sera was added to each RalA- or PBS-coated well and incubated at room temperature, while agitating at 250 rpm for 60 min. After four washes with PBS containing 0.05% Tween-20 (PBST), 100 µl horseradish peroxidase-conjugated antihuman IgG (1:5,000; Medical & Biological Laboratories Co., Ltd), diluted in 20 mM 2-[4-(2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid, 135 mM NaCl, 1% Bovine serum albumin and 0.1% hydroxiphenylacetic acid, was added to each well as a secondary antibody. The plates were incubated at room temperature, while agitating at 250 rpm for 60 min. The wells were washed four times with PBST buffer, and autoantibodies were detected by adding 100 µl of 3,3',5,5'-tetramethylbenzidine substrate. After incubation at room temperature for 30 min, the reaction was terminated by adding 0.25 N H₂SO₄ (100 µl/well). Absorbance was measured at 450 nm using the SUNRISE Microplate Reader (Tecan Japan Co., Ltd). RalA signals were determined by calculating the difference between the absorbance values for wells containing RalA and PBS. Serum carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9) markers were also evaluated as described previously (14).

Mann-Whitney U test and Fisher's exact probability test, were used to examine the differences between two groups, and Kruskal-Wallis test and Steel-Dwass test were used to compare multiple comparisons. Clinicopathological parameters associated with overall survival were evaluated by univariate analyses using log-rank test based on the Kaplan-Meier survival curves. Multivariate analyses were performed using the Cox proportional hazards model. All statistical analyses were performed using EZR (Saitama Medical Centre, Jichi Medical University; Saitama, Japan) (15), which is a graphical user interface for R (The R Foundation for Statistical Computing; version 2.13.0). P<0.05 was considered to indicate a statistically significant difference.

The optical density (mean ± standard deviation) of s-RalA-Abs was significantly higher (P<0.05) for the 291 patients with gastric cancer (0.191±0.349) compared with 73 healthy controls (0.117±0.069; Fig. 1). s-RalA-Ab levels were divided into two groups: Normal optical density values, below the cut-off level of 0.255 (calculated as mean + 2 standard deviations of the values in healthy controls) and abnormal or positive values that were 0.255 or higher. The overall positivity rate for s-RalA-Abs was 15% (43 of 291 patients).

No statistically significant differences were observed between s-RalA-Abs-positive and s-RalA-Abs-negative patients with respect to sex, age, Tumor-Node-Metastasis classification (TNM) stage (16), tumor depth and lymph node status (Table I). Moreover, no association of s-RalA-Abs with other tumor markers (CEA and CA19-9) was observed (Table I). Although the differences were not statistically significant, lower positivity rates for s-RalA-Abs was shown in T1/T2 tumors and node-positive tumors compared with others. Venous invasion was significantly associated with s-RalA-Abs (P<0.05). Lymphatic invasion and tumor size were not associated with s-RalA-Abs.

The positivity rates for various markers in patients with gastric cancer were 15% (43/291) for s-RalA-Abs, 17% (45/271) for CEA, and 14% (36/267) for CA19-9 (CEA and CA19-9 were not measured in all cases). A total of 19 patients were positive for both CEA and CA19-9, whereas only six were positive for both s-RalA-Abs and CEA, and three were positive for both s-RalA-Abs and CA19-9 (Fig. 2). Thus, the positivity rates were significantly higher for the combination assay with all the three markers [98 of 291 (34%)] compared with the two conventional markers-CEA and/or CA19-9 [62 of 291 (21%)] (P<0.01). Although the positivity rates for CEA and CA19-9 gradually increased with an increase in the tumor stage, the positivity rates for s-RalA-Abs seemed to be similar at all stages (Fig. 3). In addition, since s-RalA-Abs was independent of CEA and CA19-9, the combination of s-RalA-Abs with CEA and CA19-9 increased the detection rate for gastric cancer at each tumor stage (Fig. 4).

s-RalA-Abs-positive patients showed worse survival compared with s-RalA-Abs-negative patients, although no statistically significant differences with respect to overall survival were observed between s-RalA-Abs-positive and s-RalA-Abs-negative patients with gastric cancer (Table II; P=0.320; Fig. 5A). The patients who were triple-negative for CEA, CA19-9 and s-RalA-Abs showed significantly better overall survival compared with the other group (CEA- and/or CA19-9- and/or RalA-positive individuals) (P<0.01; Fig. 5B). By multivariate analysis, although tumor depth and lymph node status were independent risk factors for patient survival, s-RalA-Abs were not found to be independent risk factors for survival (Table II).