Prevalence of autoantibodies against Ras‑like GTPases, RalA, in patients with gastric cancer

  • Authors:
    • Tatsuki Nanami
    • Isamu Hoshino
    • Masaaki Ito
    • Satoshi Yajima
    • Yoko Oshima
    • Takashi Suzuki
    • Fumiaki Shiratori
    • Yoshihiro Nabeya
    • Kimihiko Funahashi
    • Hideaki Shimada
  • View Affiliations

  • Published online on: July 20, 2020     https://doi.org/10.3892/mco.2020.2098
  • Article Number: 28
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Abstract

Ras‑like GTPases, RalA and RalB, are members of the Ras superfamily of small GTPases. RalA expression has been shown to be associated with aggressive clinicopathological characteristics and progression in cancer. RalA protein has been shown to be involved in immune reactions in some patients with cancer; however, the clinicopathological significance of serum RalA antibody in patients with gastric cancer has not been investigated. Serum samples of 291 patients with gastric cancer and 73 healthy controls were analyzed for serum RalA antibody using enzyme‑linked immunosorbent assay. A cut‑off optical density value was fixed at 0.255 (mean of control + 2 standard deviations). The clinicopathological and prognostic significance of s‑RalA‑Abs was evaluated. The positivity rate for serum RalA antibody (s‑RalA‑Abs) was 15%. The presence of serum RalA antibody was higher in younger patients compared with elderly patients, however this tendency was not statistically significant. s‑RalA‑Abs was not associated with tumor stage. Since s‑RalA‑Abs was independent of CEA (carcinoembryonic antigen) and carbohydrate antigen 19‑9 (CA19‑9), the combination of s‑RalA‑Abs with CEA and CA19‑9 significantly increased the detection rate of gastric cancer at each tumor stage. Patients who were tested positive for s‑RalA‑Abs showed poor long‑term survival; however, this association was not statistically significant by multivariate analysis. In conclusion, s‑RalA‑Abs may be a candidate serum marker for gastric cancer, when used in combination with CEA and/or CA19‑9. Additionally, the presence of s‑RalA‑Abs, in combination with CEA and/or CA19‑9, was associated with poor survival in patients with gastric cancer.

Introduction

Ras/phosphoinositide 3-kinase (PI3K)/AKT-associated factors were one of the key molecules involved in the epithelial-to-mesenchymal transition of gastric cancer cells (1). Ras-like GTPases, RalA and RalB, are members of the Ras superfamily of small GTPases. These GTPases are aberrantly induced during tumorigenesis by oncogenic Ras (2). RalA and RalB have been reported as key cancer phenotypic markers and biomarkers of cellular migration, invasion and metastasis (3,4). RalA expression has been shown to be associated with aggressive clinicopathological characteristics and progression in squamous cell carcinoma (3,4). In gastric cancer, Ajani et al (5) reported that Gal-3 induced c-MYC expression through increased RalA activity and an enhanced YAP1/RalA/RalBP complex to confer an aggressive phenotype.

Some IgG autoantibodies have been found to respond to tumor-associated antigens in the sera of patients with cancer, even at the early stages (6,7). Since RalA is a tumor antigen, autoantibodies against RalA (s-RalA-Abs) have been reported as potential biomarkers for hepatocellular (8), esophageal (9), colorectal (10), breast (11) and ovarian (12) carcinoma. Although the role of other autoantibodies has been investigated in patients with gastric cancer (13), the significance of the clinicopathological and prognostic impact of s-RalA-Abs has not yet been demonstrated.

Therefore, the clinicopathological significance and prognostic value of preoperative s-RalA-Abs levels were evaluated in patients with gastric cancer who underwent radical surgery.

Patients and methods

Collection of sera

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).

Purification of recombinant RalA and enzyme-linked immunosorbent assay (ELISA) to detect s-RalA-Abs

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).

Statistical analyses

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.

Results

s-RalA-Ab titer

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).

Clinicopathological characteristics, conventional serum markers, and presence of s-RalA-Abs

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.

Table I

Comparisons of clinicopathological characteristics and conventional serum markers of patients with serum RalA antibodies.

Table I

Comparisons of clinicopathological characteristics and conventional serum markers of patients with serum RalA antibodies.

Variables (n)s-RalA-Abs-positive (n=43) %s-RalA-Abs-negative (n=248)P-values
Sex  0.372
     Male (201)27(13)174 
     Female (90)16(18)74 
Age, years  0.089
     <65(182)32(18)150 
     ≧65(109)11(10)98 
TNM stage  0.359
     I+II (212)34(16)178 
     III+IV (79)9(11)70 
Depth  0.774
     T1+T2(195)28(14)167 
     T3+T4(96)15(16)81 
Lymph nodea  0.125
     Negative (185)32(17)153 
     Positive (105)11(10)94 
CEAa  1
     Negative (226)33(15)193 
     Positive (45)6(13)39 
CA19-9a  0.318
     Negative (231)36(16)195 
     Positive (36)3(8)33 
Tumor sizea  0.327
     <5 cm (126)16(12)110 
     ≧5 cm (67)12(17)55 
Lya  0.520
     ly (-) (102)16(15)86 
     ly (+) (96)12(12)84 
va  0.012
     v (-) (106)21(19)85 
     v (+) (93)7(7)86 

[i] aParts of the data were not available. s-RalA-Abs, serum anti-RalA antibodies; TNM, Tumor-Node-Metastasis; CEA, carcinoembryonic antigen; CA19-9; carbohydrate antigen 19-9; Ly, lymphatic invasion; V, venous invasion.

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).

Prognostic role of s-RalA-Abs

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).

Table II

Univariate and multivariate analysis of risk factors for survival in patients with gastric cancer.

Table II

Univariate and multivariate analysis of risk factors for survival in patients with gastric cancer.

VariablesUnivariate P-valueaH.R.b95% CIcMultivariate P-valued
Sex0.705   
Age0.364   
Stage<0.001   
Tumor depth<0.00112.54.35-36.3<0.001
Nodal status<0.0011.720.716-4.130.224
s-RalA-Abs0.320   
CEA<0.001   
CA19-9<0.001   
CEA or RalA<0.012.600.802-8.450.111
CA19-9 or RalA<0.0015.241.55-17.6<0.01
CEA or CA19-9 or RalA<0.0011.821.02-3.24<0.05

[i] aLog-rank test.

[ii] bAdjusted hazards ratio.

[iii] cAdjusted 95% confidence interval.

[iv] dCox proportional hazards model. s-RalA-Abs, serum anti-RalA antibodies; CEA, carcinoembryonic antigen; CA19-9; carbohydrate antigen 19-9.

Discussion

In the present study, the positivity rate for s-RalA-Abs in patients with gastric cancer was 15%. The presence of s-RalA-Abs did not show any direct association with tumor progression. Since s-RalA-Abs were not associated with CEA or CA19-9, the combination assay increased the positivity rate. There was no significant difference in the overall survival between s-RalA-Abs-positive and s-RalA-Abs-negative patients.

Among the various clinicopathological variables, age and venous invasion seemed to be associated with the presence of s-RalA-Abs. Younger patients were more likely to produce s-RalA-Abs compared with elderly patients. This tendency was not the same as that observed in case of other autoantibodies in patients with gastric cancer (13). Since RalA expression was reported to be associated with aggressive clinicopathological characteristics and progression (3,4), s-RalA-Abs could be predictive biomarkers for poor survival. However, on univariate and multivariate analyses, s-RalA-Abs were not found to be independent risk factors for the lower overall survival. Such discrepancy can be partly explained by anti-tumoral effects of autoantibodies or may be attributable to the small sample size of patients in the present study.

The associations between TNM staging and positivity rate of various autoantibodies have been reported in hepatocellular carcinoma, colorectal cancer, breast cancer and gastric cancer (8,10,11,13). Generally, autoantibody-positive rate is relatively higher than the positive rate for conventional serum markers at stage I/II of esophageal squamous cell carcinoma (17). Among them, colorectal cancer (10) and breast cancer (11) showed that the positive rates of s-RalA-Abs were similar at all stages. A similar tendency was demonstrated in the present study in patients with gastric cancer.

A limitation of the present study was that the results of immunohistochemical examination of resected specimens were not used to evaluate the association of protein expression with s-RalA-Abs reactions. Based on a previous report, which showed the association between the presence of s-RalA-Abs and immunoreactivity (9), s-RalA-Abs may be associated with immunohistochemistry in the present study. Furthermore, a larger study evaluating the association of protein expression with s-RalA-Abs reactions may help clarify the prognostic impact of s-RalA-Abs reactions.

In conclusion, in patients with gastric cancer, the presence of s-RalA-Abs was found to be independent of other conventional serum tumor markers. s-RalA-Abs may be useful serum markers, in combination with CEA and CA19-9, for patients with gastric cancer, particularly in patients with stage I/II/III tumors. The presence of s-RalA-Abs, in combination with CEA and/or CA19-9, was also associated with poor patients' survival in gastric cancer.

Acknowledgements

The authors would like to thank Dr. Akiko Kuwajima (Medical & Biological Laboratories Co., Ltd., Nagoya, Japan) for preparing the RalA peptides.

Funding

This study was partly supported by Grant-in-Aid for Scientific Research (grant nos. 16K10519 and 16K10520) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. Research grants was obtained from the Medical & Biological Laboratories Co., Ltd. (Nagoya, Japan).

Availability of data and materials

The datasets used and/or analyzed in the present study are available from the corresponding author on reasonable request.

Authors' contributions

TN, IH, MI and HS contributed to the study conception and design. SY, YO, TS, YN, KF and FS contributed analyze the data. TN and HS analyzed patients' data and drafted the manuscript. IH revised the manuscript. All authors have read and approved the final manuscript.

Ethics approval and consent to participate

This study was approved by the institutional review boards of the Chiba Cancer Center (approval no. #21-26) and the Toho University School of Medicine (approval nos. #22-112 and #22-047). Additionally, written informed consent was obtained from all patients.

Patient consent for publication

No applicable.

Competing interests

Professor Hideaki Shimada received research grants from the Medical & Biological Laboratories Co., Ltd. (Nagoya, Japan). All other authors have no competing interests.

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October-2020
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Spandidos Publications style
Nanami T, Hoshino I, Ito M, Yajima S, Oshima Y, Suzuki T, Shiratori F, Nabeya Y, Funahashi K, Shimada H, Shimada H, et al: Prevalence of autoantibodies against Ras‑like GTPases, RalA, in patients with gastric cancer. Mol Clin Oncol 13: 28, 2020
APA
Nanami, T., Hoshino, I., Ito, M., Yajima, S., Oshima, Y., Suzuki, T. ... Shimada, H. (2020). Prevalence of autoantibodies against Ras‑like GTPases, RalA, in patients with gastric cancer. Molecular and Clinical Oncology, 13, 28. https://doi.org/10.3892/mco.2020.2098
MLA
Nanami, T., Hoshino, I., Ito, M., Yajima, S., Oshima, Y., Suzuki, T., Shiratori, F., Nabeya, Y., Funahashi, K., Shimada, H."Prevalence of autoantibodies against Ras‑like GTPases, RalA, in patients with gastric cancer". Molecular and Clinical Oncology 13.4 (2020): 28.
Chicago
Nanami, T., Hoshino, I., Ito, M., Yajima, S., Oshima, Y., Suzuki, T., Shiratori, F., Nabeya, Y., Funahashi, K., Shimada, H."Prevalence of autoantibodies against Ras‑like GTPases, RalA, in patients with gastric cancer". Molecular and Clinical Oncology 13, no. 4 (2020): 28. https://doi.org/10.3892/mco.2020.2098