Loss of viral genome with altered immune microenvironment during tumour progression of Epstein-Barr virus-associated gastric carcinoma.

Epstein-Barr virus (EBV) is one of the major drivers of gastric carcinogenesis. EBV infection is established before tumour initiation and is generally maintained throughout tumour development; however, the significance of EBV in tumour maintenance and progression remains to be elucidated. Here, we report eight cases of EBV-associated gastric carcinoma (EBVaGC) with intratumoural heterogenous expression of EBV-encoded small RNA (EBER), a highly expressed latent gene of EBV, and demonstrate clinicopathological characteristics of these rare cases. By performing detailed histological assessment of EBER-positive and -negative components of each case, detection of EBV genome in tumour cells by fluorescence in situ hybridisation, TP73 methylation analysis, whole exome sequencing, and targeted gene panel sequencing, we identified tumours in two patients to be collision tumours of different origins. In the other six patients, some genetic/epigenetic alterations were shared between EBER-positive and -negative components, suggesting that EBV was eliminated from tumour cells during progression. Interestingly, in both tumour types, programmed death ligand 1 and intratumoural infiltration of CD8+ T lymphocytes were lower in EBER-negative than in EBER-positive components, suggesting an immunogenic role of EBV. To the best of our knowledge, this study is the first to demonstrate the detailed histological features and genetic/epigenetic alterations in EBVaGC with heterogenous EBER expression; the loss of EBV may benefit tumour progression and immune evasion and might be clinically important for selecting treatment strategies for such cancers. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Somatic GJA4 gain-of-function mutation in orbital cavernous venous malformations.

Orbital cavernous venous malformation (OCVM) is a sporadic vascular anomaly of uncertain etiology characterized by abnormally dilated vascular channels. Here, we identify a somatic missense mutation, c.121G > T (p.Gly41Cys) in GJA4, which encodes a transmembrane protein that is a component of gap junctions and hemichannels in the vascular system, in OCVM tissues from 25/26 (96.2%) individuals with OCVM. GJA4 expression was detected in OCVM tissue including endothelial cells and the stroma, through immunohistochemistry. Within OCVM tissue, the mutation allele frequency was higher in endothelial cell-enriched fractions obtained using magnetic-activated cell sorting. Whole-cell voltage clamp analysis in Xenopus oocytes revealed that GJA4 c.121G > T (p.Gly41Cys) is a gain-of-function mutation that leads to the formation of a hyperactive hemichannel. Overexpression of the mutant protein in human umbilical vein endothelial cells led to a loss of cellular integrity, which was rescued by carbenoxolone, a non-specific gap junction/hemichannel inhibitor. Our data suggest that GJA4 c.121G > T (p.Gly41Cys) is a potential driver gene mutation for OCVM. We propose that hyperactive hemichannel plays a role in the development of this vascular phenotype.

Microsatellite instability profiles of gastrointestinal cancers: comparison between non-colorectal and colorectal origin.

Microsatellite instability (MSI) is a major carcinogenic pathway with prognostic and predictive implications. The validity of polymerase chain reaction (PCR)-based MSI testing is well established in colorectal cancer; however, the data are limited in non-colorectal gastrointestinal cancers. The aim of this study is to clarify the detailed MSI profiles of non-colorectal gastrointestinal cancers and to investigate the differences from those of colorectal cancers. MSI testing was performed using paired tumour/normal tissues of 123 mismatch repair-deficient cancers detected by immunohistochemistry including 80 non-colorectal cancers (eight oesophagogastric junction (EGJ), 57 gastric and 15 small intestine) and 43 colorectal cancers. Fragment size analysis revealed that the mean nucleotide shifts of five markers (Promega panel) were the highest in the stomach (6.4), followed by colorectum (5.7), small intestine (5.0) and EGJ cancers (mean = 4.0; P = 0.015, versus stomach). All cases showed ≥ 1 nucleotide shift in ≥ 2 markers and were considered as MSI-high. However, when the cut-off was set to ≥ 3 nucleotide shifts in ≥ 2 markers, three EGJ (37.5%), two small intestine (13.3%) and two gastric (3.5%) cancers showed false-negative results. In addition, cases with isolated loss of MSH6 or PMS2 showed smaller nucleotide shifts than those in others. MSI testing is applicable to non-colorectal gastrointestinal cancers; however, a subset can yield false-negative results due to subtle nucleotide shift in multiple markers. Analysis of paired tumour/normal tissues and careful interpretation is necessary to avoid false-negative results and ensure appropriate treatment.

KRAS variant allele frequency, but not mutation positivity, associates with survival of patients with pancreatic cancer.

KRAS mutation is a major driver of pancreatic carcinogenesis and will likely be a therapeutic target. Due to lack of sensitive assays for clinical samples of pancreatic cancer with low cellularity, KRAS mutations and their prognostic association have not been fully examined in large populations. In a multi-institutional cohort of 1162 pancreatic cancer patients with formalin-fixed paraffin-embedded tumor samples, we undertook droplet digital PCR (ddPCR) for KRAS codons 12/13/61. We examined detection rates of KRAS mutations by clinicopathological parameters and survival associations of KRAS mutation status. Multivariable hazard ratios (HRs) and 95% confidence intervals (CIs) for disease-free survival (DFS) and overall survival (OS) were computed using the Cox regression model with adjustment for potential confounders. KRAS mutations were detected in 1139 (98%) patients. The detection rate did not differ by age of tissue blocks, tumor cellularity, or receipt of neoadjuvant chemotherapy. KRAS mutations were not associated with DFS or OS (multivariable HR comparing KRAS-mutant to KRAS-wild-type tumors, 1.04 [95% CI, 0.62-1.75] and 1.05 [95% CI, 0.60-1.84], respectively). Among KRAS-mutant tumors, KRAS variant allele frequency (VAF) was inversely associated with DFS and OS with HRs per 20% VAF increase of 1.27 (95% CI, 1.13-1.42; ptrend <0.001) and 1.31 (95% CI, 1.16-1.48; ptrend <0.001), respectively. In summary, ddPCR detected KRAS mutations in clinical specimens of pancreatic cancer with high sensitivity irrespective of parameters potentially affecting mutation detections. KRAS VAF, but not mutation positivity, was associated with survival of pancreatic cancer patients.

KRAS mutation in intrahepatic cholangiocarcinoma: Linkage with metastasis-free survival and reduced E-cadherin expression.

BACKGROUND AND AIMS: Although KRAS mutations are the major driver of intrahepatic cholangiocarcinoma (ICC), their role remains unexplored. This study aimed to elucidate the prognostic effects, association with clinicopathologic characteristics and potent functions of KRAS mutations in ICC. METHODS: A hundred and seven resected stage I-III ICCs were analysed for KRAS mutation status and its link with clinicopathological features. An independent validation cohort (n = 138) was included. In vitro analyses using KRAS-mutant ICC cell lines were performed. RESULTS: KRAS mutation was significantly associated with worse overall survival in stage I-III ICCs, which was validated in an independent cohort. Recurrence-free survival did not significantly differ between cases with and without KRAS mutations, but if limited to recurrence with extrahepatic metastasis, KRAS-mutant cases showed significantly worse distant metastasis-free survival than KRAS-wild cases showed. KRAS mutations were associated with frequent tumour budding with reduced E-cadherin expression. In vitro, KRAS depletion caused marked inhibition of cell growth and migration together with E-cadherin upregulation in KRAS-mutant ICC cells. The RNA-sequencing assay revealed that KRAS depletion caused MYC pathway downregulation and interferon pathway upregulation. CONCLUSIONS: Our observations suggest that KRAS mutations are associated with aggressive behaviour of ICC, especially the development of extrahepatic metastasis. Mutant KRAS is likely to change the adhesive status of ICC cells, affect the responsiveness of tumour cells to interferon immune signals, and consequently promote extrahepatic metastasis. KRAS mutation status, which predicts the prognoses of patients with ICC after surgical resection, is expected to help stratify patients better for individual postoperative treatment strategies.

High-grade transformation of pancreatic neuroendocrine tumor associated with TP53 mutations: A diagnostic pitfall mimicking neuroendocrine carcinoma.

Among pancreatic neuroendocrine neoplasms, mutations in ATRX, DAXX, and MEN1 are specific to neuroendocrine tumors (NETs), whereas TP53 and RB1 mutations are characteristic of neuroendocrine carcinomas (NECs). We report a case of pancreatic NET that underwent high-grade transformation associated with acquisition of TP53 mutations. The primary pancreatic tumor consisted of conventional grade 2 NET with loss of alpha-thalassemia/mental retardation, X-linked expression and wild-type TP53, with a small focus exhibiting significant pleomorphism and increased mitotic activity of the neoplastic cells with two pathogenic TP53 mutations. Two years later, multiple liver metastases developed and were surgically resected. The metastatic tumors showed marked pleomorphism with increased mitotic activity (17/2 mm2 ) and TP53 mutations identical to the small area with TP53 mutations in the primary tumor. Liver metastases with a single TP53 mutation were also noted. Notably, hormonal phenotype has changed during progression with decreased glucagon and increased insulin expression in the metastases. Our observations suggest that TP53 mutation can occur in pancreatic NETs during progression and can be associated with phenotypic transformation. Importantly, increased pleomorphism, mitotic activity, as well as TP53 mutations could be diagnostic pitfalls leading to an overdiagnosis of NEC.

Programmed cell death protein 1/programmed death ligand 1 but not HER2 is a potential therapeutic target in gastric neuroendocrine carcinoma.

AIMS: Gastric neuroendocrine carcinoma (NEC) is a rare and aggressive subtype with a poor prognosis. We aim to investigate expression profiles of HER2 and programmed death ligand 1 (PD-L1) in gastric NEC to test the potential applicability of drugs targeting these molecules. METHODS AND RESULTS: Expression levels of HER2 and PD-L1 were evaluated in 25 gastric NECs, including 10 pure NECs and 15 mixed adenocarcinoma-NECs, and a combined positive score (CPS) was used to evaluate PD-L1 expression. The correlations of expression levels with both clinicopathological features and the expression of p53, retinoblastoma protein (Rb) and mismatch repair proteins were also analysed. Eighteen of the 25 (72%) cases showed a PD-L1 CPS of ≥ 1, which was previously shown to be associated with response to pembrolizumab. Positive nodal metastasis and low tumour-infiltrating lymphocyte (TIL) levels at the invasive margin were significantly associated with a PD-L1 CPS of < 1. The NEC component was HER2-negative in all cases, whereas HER2 positivity was observed in the adenocarcinoma component of six of 15 (40%) mixed adenocarcinoma-NECs. Mismatch repair deficiency, a mutant pattern of p53 expression and loss of Rb expression were observed in four (16%), 17 (68%) and nine (36%) cases, respectively, although these alterations were not associated with the PD-L1 CPS or other clinicopathological characteristics. CONCLUSIONS: HER2 is unlikely to be an effective target in gastric NEC owing to the lack of HER2 expression, whereas the PD-1/PD-L1 pathway is a potential therapeutic target for gastric NEC because of the relatively high prevalence of a PD-L1 CPS of ≥ 1 in this subtype.

Cancer stem cells in Epstein-Barr virus-associated gastric carcinoma.

Cancer stem cells (CSCs) play a decisive role in the development and progression of cancer. To investigate CSCs in Epstein-Barr virus (EBV)-associated carcinoma (EBVaGC), we screened previously reported stem cell markers of gastric cancer in EBV-infected gastric cancer cell lines (TMK1 and NUGC3) and identified CD44v6v9 double positive cells as candidate CSCs. CD44v6/v9+/+ cells were sorted from EBVaGC cell line (SNU719) cells and EBV-infected TMK1 cells and these cell populations showed high spheroid-forming ability and tumor formation in SCID mice compared with the respective CD44v6/v9-/- cells. Sphere-forming ability was dependent on the nuclear factor-κB (NF-κB) signaling pathway, which was confirmed by decrease of sphere formation ability under BAY 11-7082. Small interfering RNA knockdown of latent membrane protein 2A (LMP2A), one of the latent gene products of EBV infection, decreased spheroid formation in SNU719 cells. Transfection of the LMP2A gene increased the sphere-forming ability of TMK1 cells, which was mediated through NF-κB signaling. Together, these results indicate that CD44v6v9+/+ cells are CSCs in EBVaGC that are maintained through the LMP2A/NF-κB pathway. Future studies should investigate CD44v6/v9+/+ cells in normal and neoplastic gastric epithelium to prevent and treat this specific subtype of gastric cancer infected with EBV.

Oxyntic gland neoplasm of the stomach: expanding the spectrum and proposal of terminology.

Gastric neoplasms exhibiting oxyntic gland differentiation typically are composed of cells with mild cytonuclear atypia differentiating to chief cells and to a lesser extent, parietal cells. Such tumors with atypical features have been reported also and terminology for this entity remains a matter of considerable debate. We analyzed and classified 26 tumors as oxyntic gland neoplasms within mucosa (group A, eight tumors) and with submucosal invasion. The latter was divided further into those with typical histologic features (group B, 14 tumors) and atypical features, including high-grade nuclear or architectural abnormality and presence of atypical cellular differentiation (group C, four tumors). Groups A and B tumors shared similar histologic features displaying either a chief cell predominant pattern characterized by monotonous chief cell proliferation, or a well-differentiated mixed cell pattern showing admixture of chief and parietal cells resembling fundic gland. In addition, group C tumors displayed atypical cellular differentiation, including mucous neck cell and foveolar epithelium. Moderate or even marked cytological atypia was noted in group C, whereas it was usually mild in the other groups except for three group B tumors with focal moderate atypia. More than 1000 µm submucosal invasion and lymphovascular invasions were recognized only in group C. Mutation analyses identified KRAS mutation in one group C tumor as well as GNAS mutation in in one group A and group B tumors. Intramucosal tumors appear to behave biologically benign and should be classified as "oxyntic gland adenoma". Those with submucosal invasion also have low malignant potential; however, a subset will have atypical features associated with aggressive histologic features and should be designated as "adenocarcinoma of fundic gland type". Especially, we suggest "adenocarcinoma of fundic gland mucosa type" for tumors with submucosal invasion exhibiting atypical cellular differentiation, because the feature is likely to be a sign of aggressive phenotype.

Integrative analysis of genomic alterations in triple-negative breast cancer in association with homologous recombination deficiency.

Triple-negative breast cancer (TNBC) cells do not express estrogen receptors, progesterone receptors, or human epidermal growth factor receptor 2. Currently, apart from poly ADP-ribose polymerase inhibitors, there are few effective therapeutic options for this type of cancer. Here, we present comprehensive characterization of the genetic alterations in TNBC performed by high coverage whole genome sequencing together with transcriptome and whole exome sequencing. Silencing of the BRCA1 gene impaired the homologous recombination pathway in a subset of TNBCs, which exhibited similar phenotypes to tumors with BRCA1 mutations; they harbored many structural variations (SVs) with relative enrichment for tandem duplication. Clonal analysis suggested that TP53 mutations and methylation of CpG dinucleotides in the BRCA1 promoter were early events of carcinogenesis. SVs were associated with driver oncogenic events such as amplification of MYC, NOTCH2, or NOTCH3 and affected tumor suppressor genes including RB1, PTEN, and KMT2C. Furthermore, we identified putative TGFA enhancer regions. Recurrent SVs that affected the TGFA enhancer region led to enhanced expression of the TGFA oncogene that encodes one of the high affinity ligands for epidermal growth factor receptor. We also identified a variety of oncogenes that could transform 3T3 mouse fibroblasts, suggesting that individual TNBC tumors may undergo a unique driver event that can be targetable. Thus, we revealed several features of TNBC with clinically important implications.

Inflammatory Cytokines Induce Podoplanin Expression at the Tumor Invasive Front.

Tumor invasion is a critical first step in the organismic dissemination of cancer cells and the formation of metastasis in distant organs, the most important prognostic factor and the actual cause of death in most of the cancer patients. We report herein that the cell surface protein podoplanin (PDPN), a potent inducer of cancer cell invasion, is conspicuously expressed by the invasive front of squamous cell carcinomas (SCCs) of the cervix in patients and in the transgenic human papillomavirus/estrogen mouse model of cervical cancer. Laser capture microscopy combined with gene expression profiling reveals that the expression of interferon-responsive genes is up-regulated in PDPN-expressing cells at the tumor invasive front, which are exposed to CD45-positive inflammatory cells. Indeed, PDPN expression can be induced in cultured SCC cell lines by single or combined treatments with interferon-γ, transforming growth factor-ß, and/or tumor necrosis factor-α. Notably, shRNA-mediated ablation of either PDPN or STAT1 in A431 SCC cells repressed cancer cell invasion on s.c. transplantation into immunodeficient mice. The results highlight the induction of tumor cell invasion by the inflammatory cytokine-stimulated expression of PDPN in the outermost cell layers of cervical SCC.

Lipocalin-type prostaglandin D synthase-derived PGD2 attenuates malignant properties of tumor endothelial cells.

Endothelial cells (ECs) are a key component of the tumor microenvironment. They have abnormal characteristics compared to the ECs in normal tissues. Here, we found a marked increase in lipocalin-type prostaglandin D synthase (L-PGDS) mRNA (Ptgds) expression in ECs isolated from mouse melanoma. Immunostaining of mouse melanoma revealed expression of L-PGDS protein in the ECs. In situ hybridization also showed L-PGDS (PTGDS) mRNA expression in the ECs of human melanoma and oral squamous cell carcinoma. In vitro experiments showed that stimulation with tumor cell-derived IL-1 and TNF-α increased L-PGDS mRNA expression and its product prostaglandin D2 (PGD2 ) in human normal ECs. We also investigated the contribution of L-PGDS-PGD2 to tumor growth and vascularization. Systemic or EC-specific deficiency of L-PGDS accelerated the growth of melanoma in mice, whereas treatment with an agonist of the PGD2 receptor, DP1 (BW245C, 0.1 mg/kg, injected intraperitoneally twice daily), attenuated it. Morphological and in vivo studies showed that endothelial L-PGDS deficiency resulted in functional changes of tumor ECs such as accelerated vascular hyperpermeability, angiogenesis, and endothelial-to-mesenchymal transition (EndMT) in tumors, which in turn reduced tumor cell apoptosis. These observations suggest that tumor cell-derived inflammatory cytokines increase L-PGDS expression and subsequent PGD2 production in the tumor ECs. This PGD2 acts as a negative regulator of the tumorigenic changes in tumor ECs. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Gastritis-Infection-Cancer Sequence of Epstein-Barr Virus-Associated Gastric Cancer.

Epstein-Barr virus-associated gastric cancer (EBVaGC) is a representative EBV-infected epithelial neoplasm, which is now included as one of the four subtypes of The Cancer Genome Atlas molecular classification of gastric cancer. In this review, we portray a gastritis-infection-cancer sequence of EBVaGC. This virus-associated type of gastric cancer demonstrates clonal growth of EBV-infected epithelial cells within the mucosa of atrophic gastritis. Its core molecular abnormality is the EBV-specific hyper-epigenotype of CpG island promoter methylation, which induces silencing of tumor suppressor genes. This is due to the infection-induced disruption of the balance between DNA methylation and DNA demethylation activities. Abnormalities in the host cell genome, including phosphatidylinositol-4,5-biphosphate 3-kinase catalytic subunit α (PIK3CA), AT-rich interaction domain 1A (ARID1A), and programmed death-ligand 1 (PD-L1), are associated with the development and progression of EBVaGC. Furthermore, posttranscriptional modulation affects the transformation processes of EBV-infected cells, such as epithelial mesenchymal transition and anti-apoptosis, via cellular and viral microRNAs (miRNAs). Once established, cancer cells of EBVaGC remodel their microenvironment, at least partly, via the delivery of exosomes containing cellular and viral miRNAs. After exosomes are incorporated, these molecules change the functions of stromal cells, tuning the microenvironment for EBVaGC. During this series of events, EBV hijacks and uses cellular machineries, such as DNA methylation and the miRNA delivery system. This portrait of gastritis-infection-cancer sequences highlights the survival strategies of EBV in the stomach epithelial cells and may be useful for the integration of therapeutic modalities against EBV-driven gastric cancer.

Use of droplet digital PCR for quantitative and automatic analysis of the HER2 status in breast cancer patients.

PURPOSE: Digital polymerase chain reaction (dPCR) has been used to yield an absolute measure of nucleic acid concentrations. Recently, a new method referred to as droplet digital PCR (ddPCR) has gained attention as a more precise and less subjective assay to quantify DNA amplification. We demonstrated the usefulness of ddPCR to determine HER2 gene amplification of breast cancer. METHODS: In this study, we used ddPCR to measure the HER2 gene copy number in clinical formalin-fixed paraffin-embedded samples of 41 primary breast cancer patients. To improve the accuracy of ddPCR analysis, we also estimated the tumor content ratio (TCR) for each sample. RESULTS: Our determination method for HER2 gene amplification using the ddPCR ratio (ERBB2:ch17cent copy number ratio) combined with the TCR showed high consistency with the conventionally defined HER2 gene status according to ASCO-CAP (American Society of Clinical Oncology/College of American Pathologists) guidelines (P<0.0001, Fisher's exact test). The equivocal area was established by adopting 99% confidence intervals obtained by cell line assays, which made it possible to identify all conventionally HER2-positive cases with our method. In addition, we succeeded in automating a major part of the process from DNA extraction to determination of HER2 gene status. CONCLUSIONS: The introduction of ddPCR to determine the HER2 gene status in breast cancer is feasible for use in clinical practice and might complement or even replace conventional methods of examination in the future.

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.

Profiling of Virus-Encoded MicroRNAs in Epstein-Barr Virus-Associated Gastric Carcinoma and Their Roles in Gastric Carcinogenesis.

UNLABELLED: Epstein-Barr virus (EBV) is one of the major oncogenic viruses and is found in nearly 10% of gastric carcinomas. EBV is known to encode its own microRNAs (miRNAs); however, their roles have not been fully investigated. The present report is the largest series to comprehensively profile the expression of 44 known EBV miRNAs in tissue samples from patients with EBV-associated gastric carcinoma. Several miRNAs were highly expressed in EBV-associated gastric carcinoma, and in silico analysis revealed that the target genes of these EBV miRNAs had functions associated with cancer-related pathways, especially the regulation of apoptosis. Apoptosis was reduced in EBV-associated gastric carcinoma tissue samples, and gastric carcinoma cell lines infected with EBV exhibited downregulation of the proapoptotic protein Bid (the BH3-interacting domain death agonist), a member of the Bcl-2 family. The luciferase activity of the reporter vector containing the 3' untranslated region of BID was inhibited by an ebv-miR-BART4-5p mimic in gastric cancer cell lines. Transfection of an ebv-miR-BART4-5p mimic reduced Bid expression in EBV-negative cell lines, leading to reduced apoptosis under serum deprivation. The inhibition of ebv-miR-BART4-5p expression was associated with partial recovery of Bid levels in EBV-positive cell lines. The results demonstrated the antiapoptotic role of EBV miRNA via regulation of Bid expression in EBV-associated gastric carcinoma. These findings provide novel insights in the roles of EBV miRNAs in gastric carcinogenesis, which would be a potential therapeutic target. IMPORTANCE: This report is the largest series to comprehensively profile the expression of 44 known EBV miRNAs in clinical samples from EBV-associated gastric carcinoma patients. Of the EBV miRNAs, ebv-miR-BART4-5p plays an important role in gastric carcinogenesis via regulation of apoptosis.

The niche component periostin is produced by cancer-associated fibroblasts, supporting growth of gastric cancer through ERK activation.

Overexpression of periostin (POSTN), an extracellular matrix protein, has been observed in several cancers. We investigated the importance of POSTN in gastric cancer. Genome-wide gene expression analysis using publicly available microarray data sets revealed significantly high POSTN expression in cancer tissues from stage II-IV gastric cancer, compared with background normal tissues. The POSTN/vimentin mRNA expression ratio was highly associated with gene groups that regulate the cell cycle and cell proliferation. IHC showed that periglandular POSTN deposition, comprising linear deposition abutting the glandular epithelial cells in normal mucosa, disappeared during intestinal gastric cancer progression. Stromal POSTN deposition was also detected at the invasive front of intestinal-type and diffuse-type cancers. In situ hybridization confirmed POSTN mRNA in cancer-associated fibroblasts, but not in tumor cells themselves. POSTN enhanced the in vitro growth of OCUM-2MLN and OCUM-12 diffuse-type gastric cancer cell lines, accompanied by the activation of ERK. Furthermore, coinoculation of gastric cancer cells with POSTN-expressing NIH3T3 mouse fibroblast cells facilitated tumor formation. The OCUM-2MLN orthotopic inoculation model demonstrated that tumors of the gastric wall in Postn(-/-) mice were significantly smaller than those in wild-type mice. Ki-67 and p-ERK positive rates were both lower in Postn(-/-) mice. These findings suggest that POSTN produced by cancer-associated fibroblasts constitutes a growth-supportive microenvironment for gastric cancer.

PRKACA/PRKACB Fusions in Pancreatobiliary Intraductal Oncocytic Papillary Neoplasms Including Those With Atypical Morphology: An Analysis of 22 Cases Expanding Morphologic Spectrum.

Intraductal oncocytic papillary neoplasms (IOPNs) of the pancreatobiliary tract are considered a separate entity from intraductal papillary mucinous neoplasms (IPMNs), especially because of the distinct molecular alterations represented by PRKACA or PRKACB fusion. However, IOPNs display a spectrum of cytoarchitectural features. Typically, an IOPN is composed of arborizing papillae lined by layers of cells with oncocytic cytoplasm, prominent nucleoli, and intraepithelial lumina, while a significant subset shows atypical morphology: lack of the characteristic cytoarchitectural features such as arborizing papillae and prominent nucleoli, or mixture with nononcocytic IPMN-like components within a single lesion. To elucidate the tumorigenesis and morphologic spectrum of IOPNs, we analyzed 22 IOPNs, including those with atypical morphology for PRKACA/PRKACB fusions in each different component separately using fluorescence in situ hybridization. In total, 18 of 22 (82%) cases harbored PRKACA/PRKACB fusions, including 3 of 3 (100%) purely typical IOPNs and 15 of 19 (79%) IOPNs with atypical morphology. In the latter, PRKACA/PRKACB fusions were noted in atypical components as well as typical IOPN components. Notably, gastric-type IPMN-like components in the fusion-positive cases were usually low grade and had scattered neoplastic cells with eosinophilic cytoplasm, a morphologic feature suggestive of an early lesion of IOPN. In summary, most IOPNs with atypical morphology either lack characteristic cytoarchitectural features or exhibit a mixture with nononcocytic IPMN-like components, harbored PRKACA/PRKACB fusion as did typical IOPN components. Our observations expanded the morphologic spectrum of IOPNs. They are expected to be useful for correct diagnosis of this neoplasm.

Genomic and Pathologic Profiling of Very Well-Differentiated Gastric Adenocarcinoma of Intestinal Type: A Study With Emphasis on Diffuse-Type Transformation.

Very well-differentiated adenocarcinoma of intestinal type is a distinct subtype of gastric cancer characterized by anastomosing glands with a hand-in-hand pattern and low-grade cytologic atypia resembling intestinal metaplasia. This is a slow-growing neoplasm with an indolent clinical course; however, a subset demonstrates transformation into adenocarcinoma with higher-grade histology, typically diffuse-type carcinoma, and behaves aggressively. This study aimed to better characterize the genomic and pathologic features, with a focus on factors associated with diffuse-type transformation. A total of 58 cases with (n=31) and without (n=27) diffuse-type transformation were analyzed for molecular and pathologic features. First, comprehensive deep DNA sequencing was conducted in 18 cases (discovery cohort), followed by a digital droplet polymerase chain reaction of hot spot RHOA mutations in 40 cases (validation cohort). In total, RHOA mutations were the most common alteration (34%), followed by loss of ARID1A (12%), p53 alterations (10%), and CLDN18 :: ARHGAP26/6 fusions (3.4%). FGFR2 amplification was identified in an advanced case with a p53 alteration. Altered p53 expression was recognized only in higher-grade components and was significantly associated with advanced disease ( P =0.0015) and diffuse-type transformation ( P =0.026). A mixed mucin phenotype was also strongly correlated with advanced disease ( P <0.001) and diffuse-type transformation ( P <0.001). Decreased E-cadherin expression was frequently observed (74%) in poorly cohesive components. This study demonstrated that a subset of RHOA -mutant diffuse-type gastric cancers develops through the transformation of very well-differentiated adenocarcinoma of intestinal type. Our observations suggest a mixed mucin phenotype as a risk factor and alterations in p53 and E-cadherin as drivers of diffuse-type transformation.

The G protein-coupled receptor T-cell death-associated gene 8 (TDAG8) facilitates tumor development by serving as an extracellular pH sensor.

Tumors often are associated with a low extracellular pH, which induces a variety of cellular events. However, the mechanisms by which tumor cells recognize and react to the acidic environment have not been fully elucidated. T-cell death-associated gene 8 (TDAG8) is an extracellular pH-sensing G protein-coupled receptor that is overexpressed in various tumors and tumor cell lines. In this report, we show that TDAG8 on the surface of tumor cells facilitates tumor development by sensing the acidic environment. Overexpression of TDAG8 in mouse Lewis lung carcinoma (LLC) cells enhanced tumor development in animal models and rendered LLC cells resistant to acidic culture conditions by increasing activation of protein kinase A and extracellular signal-regulated kinase in vitro. Moreover, shRNA-mediated knockdown of endogenous TDAG8 in NCI-H460 human non-small cell lung cancer cells reduced cell survival in an acidic environment in vitro as well as tumor development in vivo. Microarray analyses of tumor-containing lung tissues of mice injected with TDAG8-expressing LLC cells revealed up-regulation of genes related to cell growth and glycolysis. These results support the hypothesis that TDAG8 enhances tumor development by promoting adaptation to the acidic environment to enhance cell survival/proliferation. TDAG8 may represent a therapeutic target for arresting tumor growth.