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Keywords:

  • PSCA;
  • GWAS;
  • gastric cancer;
  • survival;
  • molecular epidemiology

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Recent genome-wide association study (GWAS) has identified that the prostate stem cell antigen (PSCA) rs2294008 is involving in regulating gastric epithelial-cell proliferation, influencing the risk of diffuse-type gastric cancer. We hypothesized that PSCA rs2294008 is also associated with gastric cancer survival. We genotyped PSCA rs2294008 using TaqMan method in 943 patients with surgically resected gastric cancer. Analyses of genotype association with survival outcomes were assessed by the Kaplan-Meier method, Cox proportional hazards models and the log-rank test. There was no significant association between rs2294008 and survival of gastric cancer (log-rank p = 0.085 for CT/TT versus CC). However, in the stratification analysis of histology, we found that rs2294008 CT/TT genotypes were associated with significantly improved survival among diffuse-type gastric cancer (log-rank p = 0.025, hazard ratio [HR] = 0.75, 95% confidence interval [CI] = 0.59–0.96), compared to the CC genotype. Moreover, this protective effect was more predominant for diffuse-type gastric cancer patients with tumor size >5 cm and distant metastasis. If validated in further studies, PSCA rs2294008 could be useful marker of survival assessment and individualized clinical therapy for gastric cancer, particularly among the diffuse-type gastric cancer.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Gastric cancer is the fourth most common cancer and the second most frequent cause of cancer-related death worldwide.1 Most patients are diagnosed with advanced gastric cancer, and overall survival remains poor. Surgical resection remains the primary curative treatment option in gastric cancer with 5-year survival rates of 58–78% and 34% reported for Stage I and II disease, respectively.2 Despite this, the overall 5-year survival rate for all patients remains poor ranges between 15 and 35%.3 Although stage is the best available clinical measure of tumor aggression and prognosis, there are clearly important differences even within the same tumor stage.4 Therefore, discovery of biomarkers and their application in conjunction with traditional cancer diagnosis, staging and prognosis could to a large extent help improve early diagnosis and patient care.5 In recent years, studies have focused on the detection of genetic variants that play roles in the development and progression of gastric cancer.6

Prostate stem cell antigen (PSCA), a member of cell surface antigen, belongs to the Ly-6/Thy-1 family of glycosylphosphatidylinositol anchored surface protein.7 PSCA is largely expressed in normal prostate and overexpressed in a majority of prostate cancer, which made it a potential utility in the diagnosis and treatment of prostate cancer.8 Several other non-prostatic malignancies including bladder cancer, clear renal cell cancer, pancreatic cancer and gastric cancer were identified to express PSCA as well.9–12 It has been found that PSCA is expressed in differentiating gastric epithelial cells, has a cell-proliferation inhibition activity in vitro and is frequently silenced in gastric cancer.12 In normal stomach, PSCA is expressed at the isthmus of the gastric gland, which may harbor proliferating precursor cells, and may inhibit cell proliferation acting as a tumor suppressor.12

In a recent two-stage genome-wide association study (GWAS) of gastric cancer in Japanese populations, Sakamoto et al.12 identified a single nucleotide polymorphism (SNP) rs2294008, in Exon 1 of PSCA, associated with susceptibility to diffuse-type gastric cancer. The C to T transition of rs2294008 has been shown to reduce transcriptional activity of an upstream fragment of PSCA. Subsequently, four replication studies with meta-analysis have a significantly ascertained association between rs2294008 and gastric cancer risk in Japanese and Chinese populations.13–16 Interestingly, another GWAS conducted by Wu et al.17 further demonstrated an association of PSCA rs2294008 with bladder cancer risk, which was validated in our on-going case-control study.18 However, previous studies all focused on cancer susceptibility rather than outcomes. In this study, we hypothesized that PSCA rs2298008 is associated with survival outcomes in gastric cancer, which may be served as a prognostic marker for gastric cancer.

Material and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Study population

Our study was approved by the Institutional Review Board of Nanjing Medical University, Nanjing, China. The patients were recruited in Yixin People's Hospital, Yixin City, China. A total of 1022 gastric cancer cases were enrolled between January 1999 and December 2006, who had their surgical resection performed at the hospital. All patients were newly diagnosed without previous chemotherapy or radiotherapy before surgery. Of these, 78 patients (7.6%) were excluded because of lack of adequate information for follow-up. The maximum follow-up time was 119.0 months (last follow-up in March 2009) and the median follow-up time was 35.0 months.

Outcomes collection

Overall survival was the end point in this analysis. Date of death was obtained from inpatient and outpatient records or patients' families through follow-up telephone calls. Survival time was calculated from the date of surgery to the date of death or to the last follow-up. For gastric cancer patients, the clinicopathological variables, including tumor site, histotype, invasion, lymph node and distant metastasis status were obtained from the medical records of patients. Lauren's criteria were used to classify the tumors into intestinal-type or diffuse-type gastric cancer.19 Tumor invasion was evaluated and classified as T1, T2, T3 or T4 according to American Joint Commission for Cancer Staging (AJCCS) in 2002, the sixth edition. Lymph nodes were examined and staged according to tumor-node-metastasis (TNM) classification (AJCCS, 6th).

Genotyping

Genomic DNA was extracted from both tumor and normal tissues from the patients by proteinase K digestion, followed by isopropanol extraction and ethanol precipitation. Genotyping was performed with the TaqMan SNP Genotyping Assay using the 384-well ABI 7900HT real time PCR System (Applied Biosystems, Foster City, CA). Genotype analysis was performed by two persons independently in a blind fashion. About 10% of the samples were randomly selected for repeated genotyping for confirmation, and the results were 100% concordant. However, one case failed in genotyping because of DNA quality, which was excluded in further analyses. As a result, 943 gastric cancer cases were included in the final analysis.

Statistical analysis

The different survival times according to demographic and clinical information were estimated by using the Kaplan-Meier method and assessed by using the log-rank test. Mean survival time was presented when the median survival time (MST) could not be calculated. Univariate or multivariate Cox regression analysis was fitted to estimate the crude hazard ratios (HRs), adjusted HRs and their 95% confidence intervals (CIs), with adjustment for potential confounders. Cox stepwise regression analysis was also performed to determine predictive factors for gastric cancer prognosis, with a significance level of 0.05 for entering and 0.10 for removing the respective explanatory variables. All analyses were done with SAS (version 9.1; SAS Institute, Cary, NC) with two-sided p values.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Patient characteristics

The final population of this study consisted of 943 gastric cancer patients. The demographic characteristics and clinical information are summarized in Table 1. All patients were treated with surgical resections. In a period of up to 119.0 months of follow-up, 442 patients died. The median age was 62 years (range, 28–83 years), and there were 726 males (77.0%) and 217 females (23.0%). Tumor size, histology, depth of invasion, lymph node metastasis, distant metastasis and TNM stage were significantly associated with survival time (log-rank p < 0.05). Specifically, patients with tumor size >5 cm (MST, 48.0 months) had a 43% significantly higher risk of death (HR = 1.43, 95% CI = 1.18–1.72), compared to those with tumor size ≤ 5 cm (MST, 98.0 months). As the depth of invasion and TNM stage increased, the risk of death for gastric cancer showed a significant increase in a dose-response manner (log-rank p < 0.001). In addition, diffuse-type gastric cancer patients had a significantly higher risk of death, compared to intestinal-type gastric cancer patients (HR = 1.47, 95% CI = 1.21–1.79).

Table 1. Patients characteristics and clinical features
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Effects of PSCA rs2294008 on gastric cancer survival

As shown in Table 2, Cox regression analyses were used to assess the associations of PSCA rs2294008 genotypes with gastric cancer survival in different genetic models. There was no significant association between the genotypes and the survival of gastric cancer in any genetic models. Then, we further evaluated the association by stratified analysis of histology with intestinal-type and diffuse-type gastric cancer. As a result, rs2294008 was associated with a significantly protective effect among diffuse-type (log-rank p = 0.025, Fig. 1), but not intestinal-type (log-rank p = 0.945, Fig. 2) gastric cancer patients in a dominant model. Cox regression analyses revealed that rs2294008 CT/TT variant genotypes had a 25% significantly increased survival (HR = 0.75, 95% CI = 0.59–0.96), compared to the CC homozygote among diffuse-type gastric cancer.

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Figure 1. Overall survival of PSCA rs2294008 in diffuse-type gastric cancer patients.

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Figure 2. Overall survival of PSCA rs2294008 in intestinal-type gastric cancer patient.

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Table 2. Association between PSCA rs2294008 and gastric cancer patients' survival
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Stratified analyses among diffuse-type gastric cancer

The associations between rs2294008 and survival of diffuse-type gastric cancer patients were further evaluated by stratified analysis of tumor size, histology, depth of invasion, lymph node metastasis, distant metastasis and TNM stage. As shown in Table 3, there was no obvious evidence of differentiated association between the protective effects of rs2294008 variant genotypes and diffuse-type gastric cancer survival among different subgroups of depth of invasion, lymph node metastasis and TNM stage. However, the decreased risk was more predominant among patients with tumor size >5 cm (HR = 0.60, 95% CI = 0.42–0.86, p for heterogeneity test = 0.085) and distant metastasis (HR = 0.33, 95% CI = 0.13–0.84, p for heterogeneity test = 0.073).

Table 3. Stratified analysis of PSCA rs2294008 genotypes associated with diffuse-type gastric cancer patients' survival
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Stepwise Cox regression model for survival of diffuse-type gastric cancer

We further performed a multivariate stepwise Cox regression analysis for the effects of clinical outcomes and rs2294008 genotypes on diffuse-type gastric cancer survival. Three variables (lymph node metastasis, distant metastasis and rs2294008) were included in the regression model with a significance level of 0.05 for entering and 0.10 for removing a variable. Furthermore, when age and sex were included in the final model, the rs2294008 polymorphism was shown to be an independent protective factor for diffuse-type gastric cancer with a 24% decreased risk (HR = 0.76, 95% CI = 0.60–0.98, p = 0.031) (Table 4).

Table 4. Stepwise Cox regression analysis on diffuse-type gastric cancer patients' survival
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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

In this study with a relatively large sample size, we investigated PSCA rs2294008 with survival of gastric cancer. The results revealed that rs2294008 was associated with a significantly increased survival of diffuse-type gastric cancer in a dominant model, and this protective effect was more predominant among patients with tumor size >5 cm and distant metastasis. These findings suggested that PSCA rs2294008 may be a useful marker for predicting the prognosis of patients with diffuse-type gastric cancer.

PSCA is involved in the inhibition of cell proliferation and induction activity of cell death.20, 21 The expression of PSCA is highly abundant in gastric mucosa but is undetectable in gastric tumor tissues, suggesting that PSCA has a tumor suppressor-like character in gastric cancer. Recent GWAS in gastric cancer had shown that PSCA rs2294008 T allele was associated with risk of diffuse-type gastric cancer. It is biologically plausible in the light of the putative function of rs2294008. Sakamoto et al.12 demonstrated that substitution of the C allele with the risk allele T reduced transcriptional activity of PSCA. As a result, decreased expression of PSCA associated with the T allele may confer on individuals a higher risk of gastric cancer, which was consistent with other case-control studies.14–16 In contrast, our results found that PSCA rs2294008 T allele conferred a favorable survival for diffuse-type gastric cancer. The exact regulation mechanisms of PSCA expression and its biological function are largely unknown; therefore, the potential explanation of our finding is that the reduced PSCA expression may increase sensitivity of gastric cancer cells to chemo-/radiotherapy, thus conferring a favorable survival. Further studies are needed to address these questions.

In this study, PSCA rs2294008 was significantly associated with the survival outcomes among diffuse-type gastric cancer but not intestinal-type gastric cancer. Although the reason for the observed histology specific difference in the survival outcomes conferred by rs2294008 remains to be elucidated, this difference may be attributable to the biological differences among the histologic types of gastric cancer. The intestinal-type gastric cancer is predominates in high-risk geographic areas, especially in Japan, Korea and China, which is related to corpus-dominant gastritis with gastric atrophy and intestinal metaplasia, whereas the diffuse-type gastric cancer is more distributed geographically, which usually originates in pangastritis without atrophy.22 In addition, though the proportion of diffuse-type gastric cancer in our study was 57.3%, which was higher than that in the study reported by Lu et al., (17.8%),13 the proportion is similar to that reported in Japanese populations (60.7%).12 Our finding was also consistent with recently published GWAS, which reported that PSCA rs2294008 contribute to susceptibility to diffuse-type gastric cancer.12 Some investigators demonstrated that tumor size and distant metastasis servers as the independent prognostic factors in gastric cancer.23–25 In the stratified analyses, our results showed that the association between rs2294008 and diffuse-type gastric cancer was more predominant among patients with tumor size >5 cm and having distant metastasis, suggesting that tumor size and distant metastasis may have a joint effect with rs2294008 on survival of diffuse-type gastric cancer.

Validation of genotype-phenotype association studies requires replication using an independent group.26 Although our finding of significant associations of PSCA rs2294008 with survival outcomes was not examined in an independent sample, our finding is supported by several lines of evidence. PSCA rs2294008 has been reported to be associated with the risk of gastric cancer in four case-control studies.12, 14–16 especially in diffuse-type gastric cancer. Furthermore, our finding is biologically plausible in the light of the putative function of the polymorphism. Therefore, it is unlikely that this association occurred by chance.

It would be interesting to analyze the role of PSCA rs2294008 in the patients receiving the same protocol of anti-cancer drugs. Because our patients had received different chemotherapy drugs, we did not have the statistical power to detect drug response effects because of its small sample size. If validated as predictive markers for chemotherapy, our results, with the integration of clinical, epidemiological and genetic data, could rationalize the individualized therapy. The next step, if validated, could be clinical trials by selecting the optimal therapy according to different genotypes of the PSCA rs2294008 that would predict who will have the most benefit and better survival among gastric cancer patients.

Some limitations in the study should be addressed. First, we were able to collect data on overall survival but not disease specific survival and recurrence. The sources of survival data that we used did not reliably denote cause of death, however, most of the gastric cancer patients ultimately died of gastric-cancer-related cause. Second, although the maximum follow-up time was 119.0 months, the median follow-up time was 35.0 months in the study, which was due to more patients recruited from 2005 to 2006 (20.1% in 2005 and 20.3% in 2006). Therefore, further studies with longer follow-up time are needed to validate our findings.

In conclusion, PSCA rs2294008 was an independent prognostic marker among diffuse-type gastric cancer. Consequently, in addition to the pathologic stage, testing for the presence of rs2294008 may help identify patient subgroups at high risk for poor disease outcome, thereby helping to refine therapeutic decisions in the treatment of gastric cancer. Further validation in other independent populations and functional evaluations are needed.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This study was partly supported by National Natural Science Foundation of China (30872084 and 30972444), the Key Program for Basic Research of Jiangsu Provincial Department of Education (08KJA330001) and “Qinglan Project” Foundation for the Young Academic Leader of Jiangsu Province (to Z. Zhang).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References