Very low risk of lymph node metastasis in Epstein-Barr virus-associated early gastric carcinoma with lymphoid stroma

Between January 2006 and December 2018, 3039 gastric cancer patients were identified after a search of electronic pathology database for gastric cancer treated with gastrectomy and lymphadenectomy at the Changzhou Second People’s Hospital, which is one of tertiary medical centers in gastric cancer endemic regions in China. Over the study period, we identified 31 early gastric carcinoma patients who were treated with endoscopic submucosal dissection, which has been one of endoscopic resection methods used in our hospital since 2014. Eight of 31 (25.8%) patients also underwent subsequent gastrectomy and lymphadenectomy because of a high suspicion of LNM. Each electronic pathology report was scrutinized for the final diagnosis of early gastric carcinoma on the basis of the World Health Organization definition [2]. Excluded were synchronous gastric carcinomas, gastric stump carcinoma, and lymphoma. Micropapillary adenocarcinoma, an aggressive variant of gastric carcinomas, as described in our previous study, was also eliminated [11]. During the period from January 2010 to December 2012, 109 consecutive cases of conventional early gastric carcinomas with follow-up information were enrolled in the study as the control group for comparison, while 20 cases without follow-up information in this study period were excluded. All enrolled patients underwent D1 or D2 lymphadenectomy and all patients with EBV-GCLS underwent D2 lymphadenectomy. Conventional early gastric carcinomas were grouped into 3 subgroups, according to the invasion depth as intramucosa, superficial (SM1) or deep (SM2) submucosa, which is defined below. All patient private information was deleted and each case was coded with a pathology accession number to protect patient privacy. The study protocol was approved by the Medical Ethics Committee of the Changzhou Second People’s Hospital in China (document number: [2019] KY031–01).

The clinicopathological information on patient age, gender, tumor location, size, and gross feature was retrieved from the electronic pathology archives. The stomach was divided into 5 regions: 1) cardia, defined as the proximal gastric region of about 3 cm below the gastroesophageal junction, 2) fundus, 3) corpus, 4) incisura angularis, and 5) antrum-pylorus. Tumor macroscopic patterns were categorized into 5 groups in accordance with the Paris classification: 1) type 0-I (protruded), 2) type 0-IIa (superficial elevated), 3) type 0-IIb (flat), 4) type 0-IIc (superficial depressed) and 5) 0-III (excavated) [12]. For early gastric carcinoma with mixed macroscopic patterns, the predominant pattern was recorded. The degree of tumor penetration was assessed microscopically and tabulated into 3 subgroups: 1. intramucosa: carcinoma involvement of the lamina propria and muscularis mucosae, 2. SM1: carcinoma penetrating into superficial submucosa (up to 500 μm from the muscularis mucosae), 3. SM2: carcinoma invading into deep submucosa (beyond 500 μm from the muscularis mucosae). The tumor pathologic stage was assigned, according to the 8th edition of the American Joint Committee on Cancer staging manual [13].

All histology slides of each qualified early gastric carcinoma case were reviewed by two experienced study pathologists. The discrepancies on invasion depth and morphologic subtype were minimal and resolved by a joint review for consensus. The histological diagnostic criteria for gastric carcinoma with lymphoid stroma followed those described before [2, 14, 15]: 1) well-demarcated tumor border, 2) poorly differentiated tumor cells with indistinct cytoplasmic border in nest, sheet, anastomosing, or isolated cell growth patterns, 3) dense lymphocytic infiltration in the neoplastic epithelium and throughout the tumor stroma. EBV-GCLS was defined as early gastric carcinoma with lymphoid stroma showing positive nuclear staining for EBV-encoded RNA (EBER) by the chromogenic in situ hybridization test. Lymphovascular invasion, perineural invasion, and tumor budding were also tabulated and analyzed with the methods described in our previous study [16].

The presence of EBV in early gastric carcinomas was assessed with the EBER chromogenic in situ hybridization test. This was performed manually on unstained tumor sections in 3 μm thickness, using a probe complementary to the EBV-encoded RNA (Zhongshan Jinqiao, Beijing, China). The hybridization signal was detected with an anti-digoxigenin antibody-horseradish peroxidase conjugate and counterstained with hematoxylin. A known EBV-positive nasopharyngeal carcinoma tumor section was included in each run as the positive control. Positive staining was defined as dark brown tumor nuclear staining with negative nuclear signal in surrounding lymphocytes and non-neoplastic cells. A negative control was included in each run by eliminating the EBV-encoded RNA probe.

In 8 EBV-GCLSs and 109 consecutive cases of conventional early gastric carcinoma over the period from 2010 to 2012, patient survival investigation was carried out via a review of patient’s electronic medical archive, or telephone interview by the authors to patients or patient family members. The number of survival months after radical gastrectomy was calculated from the month of surgical resection to the month of the last follow-up interview or patient death of all causes.

The agreements in invasion depth and morphologic subtype between the two observers were almost perfect (κ = 0.92, 0.85, respectively, p < 0.05). Gastric carcinoma with lymphoid stroma was found in 11 of 595 (1.8%) qualified early gastric carcinomas, which were identified from 3039 consecutive radical gastrectomies with lymphadenectomies for gastric cancer. Eight of 11 those cases were EBV-positive (1.3%, 8/595) as the study group (Table 1). The control group consisted of 109 conventional early gastric carcinomas (carcinomas without lymphoid stroma nor micropapillary components) with the follow-up data and was further sub-grouped into intramucosal (45.9%, 50/109), SM1 (25.7%, 28/109), SM2 (28.4%, 31/109) subgroups. In the control group, 2 cases were EBV-positive by the EBER in situ hybridization test but did not have lymphoid stroma; 2 additional cases (1.8%, 2/109) were found to have synchronous non-gastric malignant tumors: one as gastrointestinal stromal tumor (pT1N0M0) located in the stomach and another as uterine leiomyoma. The average numbers of retrieved lymph nodes were 22.3, 18.1, 20.5, and 20.7 in the EBV-GCLS group, intramucosal, SM1, and SM2 subgroups of control conventional early gastric carcinomas, respectively.

In 23 cases treated only with endoscopic submucosal dissection, gastric carcinoma with lymphoid stroma was not identified. These patients showed a male-to-female ratio of 15:8 with a median age of 64 years. The macroscopic patterns II and III were identified in 20 and 3 cases, respectively. Seventeen cases arose in the gastric antrum or angularis, while 6 cases were in the gastric fundus or corpus. The median size of the tumors was 1.6 cm. The predominant (17/23, 73.9%) morphologic subtype was tubular adenocarcinoma, while papillary adenocarcinoma and poorly cohesive carcinoma were identified in 3 cases each. Submucosa invasion was found in 4 cases (3 to SM1 and 1 to SM2). No lymphovascular invasion was found.

As shown in Table 2, compared to conventional early gastric carcinomas, EBV-GCLS showed no significant differences in patient age, gender, tumor macroscopic pattern, size, lymphovascular invasion, perineural invasion, and pathological stage. However, poor tumor differentiation (100% vs 26, 25, and 35.5% respectively, p < 0.01, for intramucosal, SM1, and SM2 subgroups, respectively) and synchronous non-gastric carcinoma tumor (50% vs 2, 3.6, and 0%, respectively) were significantly more frequent in the EBV-GCLS group than in the control groups (p < 0.01). EBV-GCLS was commonly (50%, 4/8) discovered the proximal stomach (3 in the cardia and 1 in the fundus). Although the prevalence of SM2 invasion (100% vs 28.4%, p < 0.01) was significantly higher in the EBV-GCLS group than in the control group, LNM (0% vs 38.7%, p < 0.05) was significantly lower in the former than in the latter in the SM2 subgroup (Table 2).

All 8 cases of EBV-GCLS showed an expansile growth pattern with a pushing invasion front at low-power evaluation. The tumor was composed of sheets, nests of, and isolated tumor cells. The anastomosing neoplastic tubular component in the lace-like pattern was present in the lamina propria of 5 (62.5%, 5/8) tumors. Cytologically, poorly differentiated tumor cells appeared to be syncytial with indistinct cell borders, round, oval vesicular nuclei with open chromatin, and distinct nucleoli. Malignant desmoplastic reaction, frequently associated with poorly differentiated conventional carcinoma in the submucosa, was characteristically absent in all 8 EBV-GCLS cases. A minor component of the tubular growth pattern was seen in 7 (87.5%, 7/8) cases, ranging from 2 to 45% in proportion. Intra-tumoral lymphocytic infiltration was prominent in neoplastic epithelial cells and stroma (Fig. 1). The neoplastic epithelial cells showed diffuse positivity in nuclear staining with the EBER in situ hybridization test (Fig. 2). As shown in Table 1, LNM was absent in all 8 cases, and lymphovascular invasion was seen in only 1 (12.5%, 1/8) case. No tumor budding was recognized in any cases. Remarkably, four patients (50%, 4/8) with EBV-GCLS had pathologically confirmed synchronous non-gastric malignant tumors: 3 as gastrointestinal stromal tumor (pT1N0M0) in the stomach and one as clear cell renal cell carcinoma (pT1aN0M0).

The median follow-up period was 61 months (range, 18 to 126) in all 117 patients of the cohort (the study group: N = 8; and the control group: N = 109). Patients with EBV-GCLS demonstrated a higher 5-year overall survival rate (100%), compared to those with conventional early gastric carcinomas in 3 subgroups (96.5, 85.7, 83.9% for intramucosal, SM1, and SM2 subgroups, respectively). However, the difference in survival did not reach a statistically significant level because of the small sample sizes.