Viral loads correlate with upregulation of PD-L1 and worse patient prognosis in Epstein-Barr Virus-associated gastric carcinoma.

Epstein-Barr virus (EBV)-associated gastric carcinoma (EBVaGC), one of four major gastric cancer types, consists of clonal growth of EBV-infected epithelial cells. However, the significance of viral loads in each tumor cell has not been evaluated. EBV-DNA is stably maintained in episomal form in the nucleus of each cancer cell. To estimate EBV copy number per genome (EBV-CN), qPCR of viral EBNA1 and host GAPDH, standardized by Namalwa DNA (one copy/genome), was applied to the formalin-fixed paraffin embedded (FFPE) surgically resected EBVaGC specimens (n = 43) and EBVaGC cell lines (SNU-719 and NCC-24). In surgical specimens, the cancer cell ratio (CCR) was determined with image analysis, and EBV-CN was obtained by adjusting qPCR value with CCR. Fluorescent in situ hybridization (FISH) was also applied to the FFPE sections using the whole EBV-genome as a probe. In surgical specimens, EBV-CN obtained by qPCR/CCR was between 1.2 and 185 copies with a median of 9.9. EBV-CN of SNU-719 and NCC-24 was 42.0 and 1.1, respectively. A linear correlation was observed with qPCR/CCR data up to 20 copies/genome (40 signals/nucleus), the limit of FISH analysis. In addition, substantial variation in the number of EBV foci was observed. Based on qPCR/CCR, high EBV-CN (>10 copies) correlated with PD-L1 expression in cancer cells (P = 0.015), but not with other pathological indicators. Furthermore, EBVaGC with high EBV-CN showed worse disease-specific survival (P = 0.041). Our findings suggest that cancer cell viral loads may contribute to expression of the immune checkpoint molecule and promotion of cancer progression in EBVaGC.

CD47 expression in Epstein-Barr virus-associated gastric carcinoma: coexistence with tumor immunity lowering the ratio of CD8+/Foxp3+ T cells.

Epstein-Barr virus-associated gastric carcinoma (EBVaGC) frequently harbors dense lymphocytic infiltration, suggesting a specific microenvironment allowing coexistence with tumor immunity. CD47, which mediates the "do not eat me" signal in innate immunity, is also important in adaptive anti-tumor immunity. We investigated the significance of CD47 in EBVaGC compared with EBV-negative gastric cancer and the correlation with various immune cells. By immunohistochemistry of CD47, high, low, and negative expression was observed in 24, 63, and 12% of EBVaGC (n = 41), while 11, 49, and 39% of EBV-negative gastric cancer (n = 262), respectively, indicating that high expression of CD47 in cancer cells was significantly frequent and increased in EBVaGC (P = 0.043). In contrast to EBV-negative gastric carcinoma in which no significant correlation was observed between CD47 and survival, high expression of CD47 correlated significantly with worse disease-specific survival (P = 0.011) and overall survival (P = 0.013) in EBVaGC. To further clarify the role of CD47 expression in EBVaGC, digital image analysis of immune cell infiltration revealed that high CD47 expression was correlated with a lower ratio of CD8+/Foxp3+ T cells (P = 0.021), a sensitive indicator of tumor immunity. Thus, CD47 lowers anti-tumor immunity in EBVaGC by finely tuning profile of infiltrating T cells, suggesting that CD47 is an additional target for cancer immunotherapy against this virus-driven gastric cancer.

Overexpression and gene amplification of PD-L1 in cancer cells and PD-L1+ immune cells in Epstein-Barr virus-associated gastric cancer: the prognostic implications.

Cancer cells use PD-L1 to evade antitumor immunity through interaction with programmed cell death protein 1 (PD-1) on T cells. Recent whole-genome sequence studies revealed frequent gene amplification of PD-L1 in Epstein-Barr virus-associated gastric cancer (EBVaGC). To investigate the significance of PD-L1 in cancer cells and their microenvironment in EBVaGC, we studied PD-L1 expression by analysis of the public database and immunohistochemistry with fluorescent in situ hybridization (FISH) of the PD-L1 gene. Analysis of the database from The Cancer Genome Atlas also disclosed high expression of PD-L1 in EBVaGC compared with other molecular subtypes of GC. Expression of PD-L1 was frequently detected in cancer cells of EBVaGC (33/96; 34%), with infiltration of PD-L1+ immune cells in its stroma (43/96; 45%). Both expression of PD-L1 in cancer cells and PD-L1+ immune cell infiltration in EBVaGC were significantly correlated with diffuse histology according to Lauren's classification and tumor invasion (pT1b or more). As a prognostic indicator, PD-L1 expression in cancer cells correlated with poor outcomes in both overall survival and disease-specific survival (P=0.0498, 0.007). PD-L1-positive cancers had dense infiltration of PD-L1+ immune cells as well as CD8+ and PD-1+ cells in EBVaGC. FISH analysis of representative samples of the tumor demonstrated gene amplification of PD-L1 in 11% of cases. PD-L1-amplified cells corresponded to PD-L1-positive cells showing high-intensity immunohistochemical staining among cancer cells showing weak or moderate intensities. Taken together, PD-L1 expression in cancer cells and their microenvironment may contribute to the progression of EBVaGC, and gene amplification occurs as clonal evolution during progression. This specific subtype of GC infected with EBV is potentially a good candidate for immunotherapy targeting of the PD-L1/PD-1 axis.

In Epstein-Barr virus-associated gastric carcinoma a high density of CD66b-positive tumor-associated neutrophils is associated with intestinal-type histology and low frequency of lymph node metastasis.

Epstein-Barr virus-associated gastric carcinoma (EBVaGC) is characterized by prominent intratumoral lymphocyte infiltration, with some infiltration by other types of inflammatory cells, such as neutrophils. In this study, the significance of tumor-associated neutrophils (TANs) and cytotoxic T lymphocytes (CTLs) was investigated in EBVaGC. CD66b-positive TANs and CD8-positive CTLs in surgically resected EBVaGC tissues were evaluated by immunohistochemistry and digital imaging analysis. Cut-off values were determined with receiver operating characteristic curve analyses. Forty-two of 77 cases (55 %) had some infiltration of TANs (CD66b-positive areas >0.5 %, TAN(+)) and 35 (45 %) had scant infiltration (TAN(-)). Thirty-five cases (45 %) had more CTLs (CD8-positive area >18 %, CTL-high) and 42 (55 %) had fewer (CTL-low). There was no correlation between CD66b- and CD8-positive areas in the tumor (P = 0.453). TAN(+) correlated with intestinal-type histology (P = 0.048) and low frequency of lymph node metastasis (P = 0.023), while CTL-low with upper location (P = 0.030) and advanced invasion depth (pT2 or greater) (P = 0.006). Disease-specific survival was not significantly correlated with TANs or CTLs. Multivariate logistic regression analysis revealed TAN(-) to be independently associated with lymph node metastasis (P = 0.036). None of the cases of TAN(+) early EBVaGC with submucosal invasion showed lymph node metastasis (95 % confidence interval 0-13.3 %). When compared with 24 EBV-negative gastric carcinomas, EBVaGC had significantly more CTLs (P < 0.001), while there was no difference in the number of TANs. TANs in EBVaGC may suppress lymph node metastasis, and absence of TANs might suggest careful follow-up or additional therapy in cases of post-endoscopic resection.

Pulmonary Metastasis of Combined Hepatocellular and Cholangiocarcinoma: A Unique Radiographic Presentation with Air-space Consolidation Masquerading as Pneumonia and Primary Pulmonary Adenocarcinoma.

Lung metastasis showing radiographic findings of air-space consolidation is considered to be rare. This report describes the case of a man with progressive left lower lobe air-space consolidation with a history of hepatocellular carcinoma. The pulmonary lesion was initially suspected to be infection and later clinically diagnosed as primary adenocarcinoma of the lung. Although the patient was treated with systemic chemotherapy, the disease progressed very rapidly. A postmortem examination revealed that the alveolar spaces were filled with neoplastic cells subsequently proven to be metastases of combined hepatocellular and cholangiocarcinoma.