Abstract
Background: Epstein-Barr virus-associated gastric cancer (EBVaGC) is one of the four molecular subtypes of gastric cancer, as defined by the classification recently proposed by The Cancer Genome Atlas. We evaluated the correlation between EBV positivity and 18F-fluorodeoxyglucose (18F-FDG) uptake by positron emission tomography/computed tomography (PET/CT) in patients with gastric cancer. Materials and Methods: We retrospectively enrolled patients with gastric cancer who underwent pretreatment 18F-FDG PET/CT and subsequent surgical resection, and then were diagnosed with advanced gastric cancer (pathologic stage ≥T2 with any N stage). Maximum standardized uptake values (SUVmax) of gastric cancer were measured by pretreatment 18F-FDG PET/CT. EBV sequences were detected by in situ hybridization (ISH) techniques. We analyzed the correlation between EBV positivity, clinicopathologic features and metabolic activity of the primary tumor. Results: A total of 205 patients were included and 15 (7.3%) patients were identified as having EBV-positive gastric cancer. Age, gender, tumor location, and histological type showed no significant differences between EBV-positive and negative groups. EBV-positive cancer is significantly more frequent in the higher-metabolic-tumor group than in the lower one (p=0.032). The mean SUVmax of gastric cancers showed significant differences between EBV-positive and negative groups (9.9±4.2 vs. 7.0±4.8, p=0.026). Conclusion: The infection status of EBV was significantly related to the 18F-FDG uptake of primary tumors in patients with advanced gastric cancer.
Epstein-Barr virus-associated gastric cancer (EBVaGC) is defined as the monoclonal proliferation of carcinoma cells with latent EBV infection, which can be verified by in situ hybridization targeted at EBV-encoded small RNA (EBER) (1). The Cancer Genome Atlas (TCGA) research network recently proposed the following molecular classification of gastric cancer; i) Epstein-Barr virus (EBV)-positive tumors, ii) microsatellite-instable tumors, iii) genomically-stable tumors, and iv) tumors with chromosomal instability (2). Two to 20% of gastric cancers are EBV positive (3-5). In a meta-analysis of 70 studies including 15,952 cases, EBVaGCs occurred more frequently in males and non-antral regions (6). A recent large multicenter study of 4,599 gastric carcinoma cases demonstrated that EBVaGC is inversely associated with stage and EBV-positive tumor was correlated with favorable prognosis after adjusting for stage and other confounds (7).
18F-fluorodeoxyglucose (FDG) has been used in the detection of various cancers since advances in the positron emission tomography/computed tomography (PET/CT) hybrid imaging system. Gastric cancer glucose metabolic activity by 18F-FDG PET/CT is highly variable. Previous studies have shown that the metabolic activity of gastric cancer lesions is associated with depth of invasion, tumor size, lymph node metastasis, lymphovascular invasion, Lauren's classification and histologic type (8). Furthermore, several reports have studied the correlation of 18F-FDG PET/CT metabolic parameters and patient prognosis. These have demonstrated that high metabolic gastric cancers are related to poor prognosis (9-11).
To date, there has been no study investigating the relationship between metabolic activity and EBVaGC. Therefore, we evaluated the metabolic status of tumors using 18F-FDG PET/CT according to EBV positivity in patients with advanced gastric cancer.
Materials and Methods
Patients. We retrospectively enrolled patients who had confirmed gastric cancer and underwent 18F-FDG PET/CT prior to therapy followed by curative surgical resection from August 2014 to July 2016 at Seoul St. Mary's hospital. Clinicopathologic data were retrospectively reviewed. All enrolled patients underwent total or subtotal gastrectomy with D1 or D2 lymph node dissection. Tumor staging was done based on the TNM classification proposed by the Union International Cancer Control 7th edition [Leslie H. Sobin (Editor) MKGE, Christian Wittekind (Editor) (2009) TNM Classification of Malignant Tumours, 7th Edition]. Patients who had pathologic T stage 1 (pT1) were excluded from this study. This study was approved by the Institutional Review Board of Seoul St. Mary's Hospital (KC17RASI0052) and the requirement to obtain informed consent was waived.
PET/CT imaging. All patients fasted for at least 6 h before the 18F-FDG PET/CT study. 18F-FDG (3.7-4.4 MBq/kg) was injected intravenously and scanning began 60 min later. No intravenous contrast agent was used. Studies were acquired on one of the following PET/CT in-line systems: Biograph Duo, Biograph TruePoint, Biograph mCT (Siemens Medical Solutions, Knoxville, TN, USA) or Discovery 710 (GE Healthcare, Milwaukee, WI, USA). There were 6-8 bed positions, and the acquisition time was 2 min per bed position. CT began at the orbitomeatal line and progressed to the upper thigh using standard protocol: 130 kVp, 80 mAs and 5-mm slice thickness (Biograph Duo); 120 kVp, 50 mAs and 5-mm slice thickness (Biograph TruePoint); 100-120 kVp, variable mAs adjusted by topographic image and 3-mm slice thickness (Biograph mCT); and 120 kVp, variable mAs adjusted by topographic image and 2.5-mm slice thickness (Discovery 710). PET followed immediately over the same body region. The CT data were used for attenuation correction, and images were reconstructed using a standard ordered-subset expectation maximization algorithm. Point spread function correction (Biograph TruePoint, Biograph mCT, Discovery 710) and time-of-flight information (Biograph mCT, Discovery 710) were incorporated into the reconstruction algorithm.
Image analysis. All PET scans were reviewed by two experienced nuclear medicine physicians who were blinded to the histopathologic findings. PET/CT was evaluated visually and quantitatively. In the visual analysis, the PET scan was considered positive when perceptible FDG uptake that could be distinguished from physiologic gastric activity was noted at the site of the primary gastric tumor lesion as seen in the staging work-up endoscopy or enhanced CT. If the interpretations were different between the readers, the results were discussed until a consensus was reached. For quantitative analysis, SUVmax was measured from all patients by drawing a volume of interest (VOI) at the primary tumor lesion. One nuclear medicine physician (SJN) measured the metabolic parameter of the primary tumor, according to the tumor defined by consensus while blinded to the pathologic result. If no perceptible FDG uptake was noted at the tumor site, a fixed VOI was dropped at the site corresponding to the known gastric cancer, and the SUVmax was measured. All FDG PET parameters were measured using the commercial software XD3 (Mirada Medical, Oxford, UK) (12-14).
Clinicopathological factors. We assessed the relationships between the following histopathological variables: depth of tumor invasion (pT stage), location of tumor, nodal metastasis (pN stage), and histologic type. pT and pN stages were based on the TNM 7th edition. Tumor location was categorized into antral or non-antral regions. The histopathologic type at the primary tumor site was categorized as papillary adenocarcinoma, tubular adenocarcinoma (well or moderately differentiated), poorly differentiated adenocarcinoma, signet-ring cell carcinoma or mucinous adenocarcinoma according to the World Health Organization classification with Japanese modification (15, 16). To detect EBV, the Epstein-Barr virus-encoded small RNA-1 (EBER1)-in situ hybridization method was used.
Statistical analysis. Continuous variables are expressed as means (±standard deviation) or medians (range) and were compared using independent t-tests or ANOVA. To evaluate the relationship between two categorical variables, we used crosstab. The statistical analysis was performed using SPSS software version 18.0 (IBM, Armonk, NY, USA). All p-values were two-sided, and a p<0.05 was considered statistically significant.
Results
Patient characteristics. A total of 463 consecutive patients underwent 18F-FDG PET/CT followed by surgery for gastric cancer. Among them, 256 patients who were diagnosed as pathologic T1 were excluded from the study. After two patients were excluded due to lack of EBV ISH, a total 205 patients (146 males, 59 females) were enrolled. The patient group mean age was 63.1±13.6 years. The patient characteristics are summarized in Table I.
Relationship between SUVmax and clinicopathologic characteristics. The mean of SUVmax was 7.2±4.8 (median=5.5; range=1.7-26.0). Patients younger than 65 years showed significantly lower metabolic activity than those 65 years and older (p=0.005, Table II). Large tumors (≥6 cm) demonstrated significantly higher SUVmax than small tumors (<6 cm) (p<0.001). Tumor SUVmax was significantly increased according to depth of invasion (p=0.005). Papillary/tubular adenocarcinoma and poorly differentiated adenocarcinoma showed significantly higher metabolic activity than signet ring cell carcinoma or mucinous adenocarcinoma (p<0.001). There were no statistically significant differences in SUVmax according to gender, lymph node metastasis and tumor location.
Patients characteristics.
Relationship between EBV positivity and clinicopathologic characteristics. Fifteen out of 205 patients showed EBV positivity (7.3%). Gastric cancers with EBV positivity were more frequent in the higher metabolic group (SUVmax ≥5.5) than lower metabolic group (SUVmax <5.5) (13/103 vs. 2/102, p=0.006, Table III). EBV-positive gastric cancers showed significantly higher SUVmax than EBV-negative cancers (mean±SD, 9.9±4.2 vs. 7.0±4.8, p=0.026, Figure 1 and Table II). EBV positivity showed no significant correlation with age, gender, tumor size, depth of invasion, lymph node metastasis, or tumor location.
Discussion
EBV-associated gastric cancer type differs from other gastric cancers, exhibiting male predominance, non-antral anatomic subsites and better survival (6, 7, 17, 18). Our results showed that EBVaGC was significantly more frequent in the higher metabolic tumor group than the lower metabolic group. To our knowledge, this is the first study to analyze the association between EBV positivity and 18F-FDG uptake in patients with gastric cancer.
In our cohort, 13 of 146 (8.9%) males showed EBV positivity and 3.4% (2/59) of females were positive. The EBV positivity according to the location was 11.2% (11/87) in non-antral regions and 3.9% (4/103) in antral regions. These predominances are well-established from previous studies. However, there were no statistically significant differences in the current study, possibly because of the small number of EBV-positive patients.
Relationship between SUVmax and clinicopathologic characteristics.
Relationship between EBV positivity and clinicopathologic characteristics.
Box plots of the SUVmax from gastric cancers according to the EBV infection status.
The frequency of EBV positivity was statistically different in the two groups for SUVmax divided by median. EBVaGCs were more frequently observed in the higher metabolic group. The reason why EBVaGC was correlated with 18F-FDG uptake is unknown. However, one possible explanation could be dysregulation of one or more metabolic signaling pathways. Previous studies have shown that PIK3CA, the gene encoding the catalytic subunit p110 of PI3K, is mutated in EBV-positive gastric cancer (2, 19). This genetic aberration results in dysregulation of the phosphatidylinositol-3 kinases (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway (20). A study in lung cancer showed that FDG uptake was associated with the molecules relevant to this signaling pathway (21). Recently Zhang et al. provided evidence that EBV latent membrane protein 1 (LMP1) upregulated Glut-1 transcription to control aerobic glycolysis and tumorigenic growth of nasopharyngeal cancer cells through mTORC1/NF-ĸB signaling (22).
Prominent inflammatory infiltration within the tumor is one of the distinct features of EBVaGC (23). These tumor-infiltrating cells are primarily lymphocytic, particularly CD8-positive or CD4-positive T cells accompanied by CD68-positive histiocytes (24-26). This feature reflects the immunogenicity of EBV, and this immune response by the host could be one reason for a better prognosis (18). The fact that increased 18F-FDG uptake has been observed in metabolically-active inflammatory cells (27) could be one explanation for the relationship of 18F-FDG uptake in EBVaGC. A recent study presented the association between metabolic parameters on 18F-FDG-PET and intra-tumor expression of immune-related markers in patients with non-small cell lung cancer (28).
Several previous studies have examined the correlation between 18F-FDG uptake and histopathological features in patients with gastric cancer. Our results showed tumor size, depth of invasion and histologic types were related to tumor 18F-FDG uptake, consistent with results from previous studies.
There existed several limitations in the current study. First, this study included a small number of patients. EBVaGC is a relatively rare type of gastric cancer and only 15 of 205 (7%) was EBV positive in our cohort. Further study with larger number of patients is needed to validate our findings. However, to our knowledge, this is the first study to evaluate the relationship between metabolic activity using 18F-FDG PET/CT and EBV positivity. Second, we enrolled patients with T2 and more severe T stage after surgical resection. Therefore, it will be hard to generalize these findings to all stages of gastric cancer.
Previous studies showing the possibility of 18F-FDG PET/CT as an imaging biomarker in stomach cancer patients have reported that patients with high FDG uptake generally have a poor prognosis (29). Our study showed that EBVaGC, which is known to have good prognosis, is associated with higher metabolic activity. To explain this finding, further studies are required to further analyze the information on tumor-infiltrating immune cells or gene mutations. Based on this association, molecular classification of gastric cancer would be considered when studying the role of PET as an imaging biomarker.
In conclusion, our preliminary findings showed that EBV positivity related to 18F-FDG uptake and high 18F-FDG activity was more frequent in patients with advanced gastric cancer. Further studies are needed to validate these findings in a larger patient study and clarify the relationship between EBV positivity and FDG uptake.
Footnotes
This article is freely accessible online.
- Received March 22, 2017.
- Revision received May 18, 2017.
- Accepted May 27, 2017.
- Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
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