Expression patterns of HNF4α, TTF-1, and SMARCA4 in lung adenocarcinomas: impacts on clinicopathological and genetic features.

INTRODUCTION: HNF4α expression and SMARCA4 loss were thought to be features of non-terminal respiratory unit (TRU)-type lung adenocarcinomas, but their relationships remained unclear. MATERIALS AND METHODS: HNF4α-positive cases among 241 lung adenocarcinomas were stratified based on TTF-1 and SMARCA4 expressions, histological subtypes, and driver mutations. Immunohistochemical analysis was performed using xenograft tumors of lung adenocarcinoma cell lines with high HNF4A expression. RESULT: HNF4α-positive adenocarcinomas(n = 33) were divided into two groups: the variant group(15 mucinous, 2 enteric, and 1 colloid), where SMARCA4 was retained in all cases, and the conventional non-mucinous group(6 papillary, 5 solid, and 4 acinar), where SMARCA4 was lost in 3/15 cases(20%). All variant cases were negative for TTF-1 and showed wild-type EGFR and frequent KRAS mutations(10/18, 56%). The non-mucinous group was further divided into two groups: TRU-type(n = 7), which was positive for TTF-1 and showed predominantly papillary histology(6/7, 86%) and EGFR mutations(3/7, 43%), and non-TRU-type(n = 8), which was negative for TTF-1, showed frequent loss of SMARCA4(2/8, 25%) and predominantly solid histology(4/8, 50%), and never harbored EGFR mutations. Survival analysis of 230 cases based on histological grading and HNF4α expression revealed that HNF4α-positive poorly differentiated (grade 3) adenocarcinoma showed the worst prognosis. Among 39 cell lines, A549 showed the highest level of HNF4A, immunohistochemically HNF4α expression positive and SMARCA4 lost, and exhibited non-mucinous, high-grade morphology in xenograft tumors. CONCLUSION: HNF4α-positive non-mucinous adenocarcinomas included TRU-type and non-TRU-type cases; the latter tended to exhibit the high-grade phenotype with frequent loss of SMARCA4, and A549 was a representative cell line.

Artificial intelligence for evaluating the risk of gastric cancer: reliable detection and scoring of intestinal metaplasia with deep learning algorithms.

BACKGROUND AND AIMS: Gastric cancer (GC) is associated with chronic gastritis. To evaluate the risk, the Operative Link on Gastric Intestinal Metaplasia Assessment (OLGIM) system was constructed and showed a higher GC risk in stage III or IV patients, determined by the degree of intestinal metaplasia (IM). Although the OLGIM system is useful, evaluating the degree of IM requires substantial experience to produce precise scoring. Whole-slide imaging is becoming routine, but most artificial intelligence (AI) systems in pathology are focused on neoplastic lesions. METHODS: Hematoxylin and eosin-stained slides were scanned. Images were divided into each gastric biopsy tissue sample and labeled with an IM score. IM was scored as follows: 0 (no IM), 1 (mild IM), 2 (moderate IM), and 3 (severe IM). Overall, 5753 images were prepared. A deep convolutional neural network (DCNN) model, ResNet50, was used for classification. RESULTS: ResNet50 classified images with and without IM with a sensitivity of 97.7% and specificity of 94.6%. IM scores 2 and 3, involved as criteria of stage III or IV in the OLGIM system, were classified by ResNet50 in 18%. The respective sensitivity and specificity values of classifying IM between scores 0 and 1 and 2 and 3 were 98.5% and 94.9%, respectively. The IM scores classified by pathologists and the AI system were different in only 438 images (7.6%), and we found that ResNet50 tended to miss small foci of IM but successfully identified minimal IM areas that pathologists missed during the review. CONCLUSIONS: Our findings suggested that this AI system would contribute to evaluating the risk of GC accuracy, reliability, and repeatability with worldwide standardization.

Lost expression of AT-rich interaction domain 1A in the gastric mucosa-A constituent of field cancerization in the stomach.

Abnormalities of the AT-rich interaction domain 1A (ARID1A) occur in cancer tissues and precursors or premalignant lesions in various organs. To investigate the significance of ARID1A abnormalities in the early phase of cancer development in the stomach, we screened for ARID1A loss and p53 overexpression in glands in non-neoplastic gastric mucosa using immunohistochemistry. We tested 230 tissue blocks of 77 patients with gastric carcinoma, and in 10% of non-neoplastic mucosa we detected ARID1A-lost and in 3.7% p53-overexpressed foci. Loss of ARID1A expression occurred in the scales of several glands, which were morphologically characterized as authentic, pseudo-pyloric, or intestinal metaplastic glands devoid of dysplastic changes. In contrast, p53-overexpressed foci were detected in dysplastic intestinal metaplasia. In early gastric cancer cases (n = 46), ARID1A-lost foci were frequent in samples of patients with Epstein-Barr virus-associated gastric carcinoma (p = 0.037). Ultra-deep DNA sequencing of ARID1A-lost foci revealed frameshift and nonsense mutations in ARID1A. Mapping abnormal glands in the entire resected stomach of the three selected patients demonstrated that ARID1A-lost foci clustered with p53 abnormal glands. ARID1A-lost epithelial cells may develop clonal growth through the pathway, different from p53-abnormal intestinal metaplasia, and require one or more events to develop into an overt carcinoma, such as EBV infection.

Adenocarcinoma of the stomach and esophagogastric junction with low DNA methylation show poor prognoses.

BACKGROUND: Gastric cancer (GC) is characterized by unique DNA methylation epigenotypes (MEs). However, MEs including adenocarcinomas of the esophagogastric junction (AEG) and background non-neoplastic columnar mucosae (NM) remain to be clarified. METHODS: We analyzed the genome-wide DNA MEs of AEG, GC, and background NM using the Infinium 450 k beadarray, followed by quantitative pyrosequencing validation. Large-scale data from The Cancer Genome Atlas (TCGA) were also reviewed. RESULTS: Unsupervised two-way hierarchical clustering using Infinium data of 21 AEG, 30 GC, and 11 NM revealed four DNA MEs: extremely high-ME (E-HME), high-ME (HME), low-ME (LME), and extremely low-ME (E-LME). Promoter methylation levels were validated by pyrosequencing in 146 samples. Non-inflammatory normal mucosae were clustered into E-LME, whereas gastric or esophagogastric junction mucosae with chronic inflammatory changes caused by either Helicobacter pylori infection or reflux esophagitis were clustered together into LME, suggesting that inflammation status determined DNA MEs regardless of the cause. Three cases of Barrett's-related adenocarcinoma were clustered into HME. Among 94 patients whose tumors could be clustered into one of four MEs, 11 patients with E-LME cancers showed significantly shorter overall survival than that in the other MEs, even with the multivariate Cox regression estimate. TCGA data also showed enrichment of AEG in HME and a poorer prognosis in E-LME. CONCLUSIONS: E-LME cases, newly confirmed in this study, form a unique subtype with poor prognosis that is not associated with inflammation-associated elevation of DNA methylation levels. LME could be acquired via chronic inflammation, regardless of the cause, and AEG might preferentially show HME.

Genetic and phenotypic determinants of morphologies in 3D cultures and xenografts of lung tumor cell lines.

We previously proposed the classification of lung adenocarcinoma into two groups: the bronchial epithelial phenotype (BE phenotype) with high-level expressions of bronchial epithelial markers and actionable genetic abnormalities of tyrosine kinase receptors and the non-BE phenotype with low-level expressions of bronchial Bronchial epithelial (BE) epithelial markers and no actionable genetic abnormalities of tyrosine kinase receptors. Here, we performed a comprehensive analysis of tumor morphologies in 3D cultures and xenografts across a panel of lung cancer cell lines. First, we demonstrated that 40 lung cancer cell lines (23 BE and 17 non-BE) can be classified into three groups based on morphologies in 3D cultures on Matrigel: round (n = 31), stellate (n = 5), and grape-like (n = 4). The latter two morphologies were significantly frequent in the non-BE phenotype (1/23 BE, 8/17 non-BE, p = 0.0014), and the stellate morphology was only found in the non-BE phenotype. SMARCA4 mutations were significantly frequent in stellate-shaped cells (4/4 stellate, 4/34 non-stellate, p = 0.0001). Next, from the 40 cell lines, we successfully established 28 xenograft tumors (18 BE and 10 non-BE) in NOD/SCID mice and classified histological patterns of the xenograft tumors into three groups: solid (n = 20), small nests in desmoplasia (n = 4), and acinar/papillary (n = 4). The latter two patterns were characteristically found in the BE phenotype. The non-BE phenotype exhibited a solid pattern with significantly less content of alpha-SMA-positive fibroblasts (p = 0.0004) and collagen (p = 0.0006) than the BE phenotype. Thus, the morphology of the tumors in 3D cultures and xenografts, including stroma genesis, reflects the intrinsic properties of the cancer cell lines. Furthermore, this study serves as an excellent resource for lung adenocarcinoma cell lines, with clinically relevant information on molecular and morphological characteristics and drug sensitivity.

Derivation of pancreatic acinar cell carcinoma cell line HS-1 as a patient-derived tumor organoid.

Acinar cell carcinoma (ACC) of the pancreas is a malignant tumor of the exocrine cell lineage with a poor prognosis. Due to its rare incidence and technical difficulties, few authentic human cell lines are currently available, hampering detailed investigations of ACC. Therefore, we applied the organoid culture technique to various types of specimens, such as bile, biopsy, and resected tumor, obtained from a single ACC patient. Despite the initial propagation, none of these organoids achieved long-term proliferation or tolerated cryopreservation, confirming the challenging nature of establishing ACC cell lines. Nevertheless, the biopsy-derived early passage organoid developed subcutaneous tumors in immunodeficient mice. The xenograft tumor histologically resembled the original tumor and gave rise to infinitely propagating organoids with solid features and high levels of trypsin secretion. Moreover, the organoid stained positive for carboxylic ester hydrolase, a specific ACC marker, but negative for the duct cell marker CD133 and the endocrine lineage marker synaptophysin. Hence, we concluded the derivation of a novel ACC cell line of the pure exocrine lineage, designated HS-1. Genomic analysis revealed extensive copy number alterations and mutations in EP400 in the original tumor, which were enriched in primary organoids. HS-1 displayed homozygous deletion of CDKN2A, which might underlie xenograft formation from organoids. Although resistant to standard cytotoxic agents, the cell line was highly sensitive to the proteasome inhibitor bortezomib, as revealed by an in vitro drug screen and in vivo validation. In summary, we document a novel ACC cell line, which could be useful for ACC studies in the future.

Assessment of liver function-related mRNA expression and fluorescence imaging in outflow-obstructed regions in rats.

PURPOSE: The liver function in outflow-obstructed regions is reportedly impaired; however, the functional decrease has not been quantitatively assessed. We therefore evaluated the uptake of indocyanine green (ICG) into hepatocytes and the mRNA expression associated with the liver function in outflow-obstructed regions using rat models. METHODS: A total of 20 rats with the ligation of the right median hepatic vein to induce outflow obstruction were studied. Five rats each were grouped by the time of re-laparotomy, and the fluorescence intensity (FI) values of ICG. The mRNA expression, including that of Albumin, Cytochrome P450 (Cyp) 1a2, Cyp3a1, Cyp7a1, and Gamma-glutamylcysteine synthetase, in outflow-obstructed (mRNAOut-Ob) and non-outflow-obstructed (mRNANon) regions was assessed. RESULTS: Microscopic fluorescence imaging showed that the FI values were significantly lower in outflow-obstructed regions than in non-outflow-obstructed regions at 12 h, 24 h, and 3 days after ligation of the hepatic vein. The mRNAOut-Ob/mRNANon ratios decreased to approximately 30% at 12 h after the outflow obstruction and increased to approximately 70-80% at 7 days. CONCLUSIONS: The liver function in outflow-obstructed regions was impaired in terms of the uptake of ICG and the mRNA expression. Our findings may help estimate the postoperative functional remnant liver volume by considering the decrease in the liver function in outflow-obstructed regions.

Integrated environmental, lifestyle, and epigenetic risk prediction of primary gastric neoplasia using the longitudinally monitored cohorts.

BACKGROUND: DNA methylation accumulates in non-malignant gastric mucosa after exposure to pathogens. To elucidate how environmental, methylation, and lifestyle factors interplay to influence primary gastric neoplasia (GN) risk, we analyzed longitudinally monitored cohorts in Japan and Singapore. METHODS: Asymptomatic subjects who underwent a gastric mucosal biopsy on the health check-up were enrolled. We analyzed the association between clinical factors and GN development using Cox hazard models. We further conducted comprehensive methylation analysis on selected tissues, including (i) mucosae from subjects developing GN later, (ii) mucosae from subjects not developing GN later, and (iii) GN tissues and surrounding mucosae. We also use the methylation data of mucosa collected in Singapore. The association between methylation and GN risk, as well as lifestyle and methylation, were analyzed. FINDINGS: Among 4234 subjects, GN was developed in 77 subjects. GN incidence was correlated with age, drinking, smoking, and Helicobacter pylori (HP) status. Accumulation of methylation in biopsied gastric mucosae was predictive of higher future GN risk and shorter duration to GN incidence. Whereas methylation levels were associated with HP positivity, lifestyle, and morphological alterations, DNA methylation remained an independent GN risk factor through multivariable analyses. Pro-carcinogenic epigenetic alterations initiated by HP exposure were amplified by unfavorable but modifiable lifestyle choices. Adding DNA methylation to the model with clinical factors improved the predictive ability for the GN risk. INTERPRETATION: The integration of environmental, lifestyle, and epigenetic information can provide increased resolution in the stratification of primary GN risk. FUNDING: The funds are listed in Acknowledgements section.

N-terminal peptide fragment constitutes core of amyloid deposition of serum amyloid A: An imaging mass spectrometry study.

Serum amyloid A (SAA) is an acute phase protein, which undergoes structural changes and deposits in the extracellular matrix, causing organ damage. Systemic AA amyloidosis is a relatively common amyloid subtype among the more than 30 amyloid subtypes, but the mechanism of amyloid fibril formation remains unclear. In this study, we investigated the tissue distribution of SAA derived peptides in formalin-fixed paraffin embedded (FFPE) specimens of human myocardium with amyloidosis using matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS). In the whole SAA protein, four trypsin-digested peptides in the range of SAA2-67 were visualized and the N-terminal peptide; SAA2-15, was selectively localized in the Congo red-positive region. The C-terminal peptides; SAA47-62, SAA48-62, and SAA63-67 were detected not only in the Congo red-positive region but also in the surrounding negative region. Our results demonstrate that the N-terminal SAA2-15 plays a critical role in the formation of AA amyloid fibril, as previously reported. Roles of the C-terminal peptides require further investigation.

Development and multi-institutional validation of an artificial intelligence-based diagnostic system for gastric biopsy.

To overcome the increasing burden on pathologists in diagnosing gastric biopsies, we developed an artificial intelligence-based system for the pathological diagnosis of gastric biopsies (AI-G), which is expected to work well in daily clinical practice in multiple institutes. The multistage semantic segmentation for pathology (MSP) method utilizes the distribution of feature values extracted from patches of whole-slide images (WSI) like pathologists' "low-power view" information of microscopy. The training dataset included WSIs of 4511 gastric biopsy tissues from 984 patients. In tissue-level validation, MSP AI-G showed better accuracy (91.0%) than that of conventional patch-based AI-G (PB AI-G) (89.8%). Importantly, MSP AI-G unanimously achieved higher accuracy rates (0.946 ± 0.023) than PB AI-G (0.861 ± 0.078) in tissue-level analysis, when applied to the cohorts of 10 different institutes (3450 samples of 1772 patients in all institutes, 198-555 samples of 143-206 patients in each institute). MSP AI-G had high diagnostic accuracy and robustness in multi-institutions. When pathologists selectively review specimens in which pathologist's diagnosis and AI prediction are discordant, the requirement of a secondary review process is significantly less compared with reviewing all specimens by another pathologist.

Universal encoding of pan-cancer histology by deep texture representations.

Cancer histological images contain rich biological and clinical information, but quantitative representation can be problematic and has prevented the direct comparison and accumulation of large-scale datasets. Here, we show successful universal encoding of cancer histology by deep texture representations (DTRs) produced by a bilinear convolutional neural network. DTR-based, unsupervised histological profiling, which captures the morphological diversity, is applied to cancer biopsies and reveals relationships between histologic characteristics and the response to immune checkpoint inhibitors (ICIs). Content-based image retrieval based on DTRs enables the quick retrieval of histologically similar images using The Cancer Genome Atlas (TCGA) dataset. Furthermore, via comprehensive comparisons with driver and clinically actionable gene mutations, we successfully predict 309 combinations of genomic features and cancer types from hematoxylin-and-eosin-stained images. With its mounting capabilities on accessible devices, such as smartphones, universal encoding for cancer histology has a strong impact on global equalization for cancer diagnosis and therapies.

A design of forceps-type coincidence radiation detector for intraoperative LN diagnosis: clinical impact estimated from LNs data of 20 esophageal cancer patients.

PURPOSE: To reduce postoperative complications, intraoperative lymph node (LN) diagnosis with 18F-fluoro-2-deoxy-D-glucose (FDG) is expected to optimize the extent of LN dissection, leading to less invasive surgery. However, such a diagnostic device has not yet been realized. We proposed the concept of coincidence detection wherein a pair of scintillation crystals formed the head of the forceps. To estimate the clinical impact of this detector, we determined the cut-off value using FDG as a marker for intraoperative LN diagnosis in patients with esophageal cancer, the specifications needed for the detector, and its feasibility using numerical simulation. METHODS: We investigated the dataset including pathological diagnosis and radioactivity of 1073 LNs resected from 20 patients who underwent FDG-positron emission tomography followed by surgery for esophageal cancer on the same day. The specifications for the detector were determined assuming that it should measure 100 counts (less than 10% statistical error) or more within the intraoperative measurement time of 30 s. The detector sensitivity was estimated using GEANT4 simulation and the expected diagnostic ability was calculated. RESULTS: The cut-off value was 620 Bq for intraoperative LN diagnosis. The simulation study showed that the detector had a radiation detection sensitivity of 0.96%, which was better than the estimated specification needed for the detector. Among the 1035 non-metastatic LNs, 815 were below the cut-off value. CONCLUSION: The forceps-type coincidence detector can provide sufficient sensitivity for intraoperative LN diagnosis. Approximately 80% of the prophylactic LN dissections in esophageal cancer can be avoided using this detector.

Virus-host interactions in carcinogenesis of Epstein-Barr virus-associated gastric carcinoma: Potential roles of lost ARID1A expression in its early stage.

Epstein-Barr virus (EBV)-associated gastric carcinoma (EBVaGC) is a distinct molecular subtype of gastric cancer characterized by viral infection and cellular abnormalities, including loss of AT-rich interaction domain 1A (ARID1A) expression (lost ARID1A). To evaluate the significance of lost ARID1A in the development of EBVaGC, we performed in situ hybridization of EBV-encoded RNA (EBER) and immunohistochemistry of ARID1A in the non-neoplastic gastric mucosa and intramucosal cancer tissue of EBVaGC with in vitro infection analysis of ARID1A-knockdown and -knockout gastric cells. Screening of EBER by in situ hybridization revealed a frequency of approximately 0.2% EBER-positive epithelial cells in non-neoplastic gastric mucosa tissue samples. Six small foci of EBV-infected epithelial cells showed two types of histology: degenerated (n = 3) and metaplastic (n = 3) epithelial cells. ARID1A was lost in the former type. In intramucosal EBVaGC, there were ARID1A-lost (n = 5) and -preserved tumors (n = 7), suggesting that ARID1A-lost carcinomas are derived from ARID1A-lost precursor cells in the non-neoplastic mucosa. Lost ARID1A was also observed in non-neoplastic mucosa adjacent to an ARID1A-lost EBVaGC. In vitro experiments using siRNA knockdown and the CRISPR/Cas9-knockout system demonstrated that transient reduction or permanent loss of ARID1A expression markedly increased the efficiency of EBV infection to stomach epithelial cells. Taken together, lost ARID1A plays a role in initiating EBV-driven carcinogenesis in stomach epithelial cells, which develop to a distinct subtype of EBVaGC within the proper mucosal layer. Lost ARID1A is one of the constituents of virus-host interactions in the carcinogenesis of EBVaGC.

Human Leukocyte Antigen Class I Deficiency in Gastric Carcinoma: An Adaptive Immune Evasion Strategy Most Common in Microsatellite Instable Tumors.

Immune checkpoint inhibitor therapy is effective only for a subset of patients with gastric cancer. Impaired neoantigen presentation caused by deficiency of human leukocyte antigen class I (HLA-I) has been reported as a common mechanism of immune evasion which is associated with resistance to immune checkpoint blockade. To elucidate the significance of HLA-I deficiency in gastric cancer with special focus on microsatellite instable (MSI) and Epstein-Barr virus (EBV)-positive tumors, we examined HLA-I expression on tumor cells and correlated the results with clinicopathologic features, programmed death-ligand 1 (PD-L1) expression, and degree of tumor-infiltrating immune cells. This study included 58 MSI, 44 EBV-positive, and 107 non-EBV non-MSI tumors for comparison. The frequency of HLA-I deficiency (≥1% tumor cells) was significantly higher in MSI tumors (52%) compared with EBV-positive tumors (23%) and the other tumors (28%). In contrast, PD-L1 expression levels were highest in EBV-positive tumors, followed by MSI tumors, with the lowest prevalence in the other tumors in both Tumor Proportion Score and Combined Positive Score. HLA-I deficiency was significantly more frequent in advanced tumors (pT2-4) than in early tumors (pT1) in MSI and non-EBV non-MSI subtypes. In addition, the degree of CD8-positive cells infiltration was significantly reduced in HLA-I deficient tumor areas compared with HLA-I preserved tumor area within a tumor. Based on our observations, HLA-I, as well as PD-L1, should be considered as a common mechanism of immune escape especially in the MSI subtype, and therefore could be a biomarker predicting response to immune checkpoint inhibitor therapy in gastric cancer.

Molecular classification and diagnostics of upper urinary tract urothelial carcinoma.

Upper urinary tract urothelial carcinoma (UTUC) is one of the common urothelial cancers. Its molecular pathogenesis, however, is poorly understood, with no useful biomarkers available for accurate diagnosis and molecular classification. Through an integrated genetic study involving 199 UTUC samples, we delineate the landscape of genetic alterations in UTUC enabling genetic/molecular classification. According to the mutational status of TP53, MDM2, RAS, and FGFR3, UTUC is classified into five subtypes having discrete profiles of gene expression, tumor location/histology, and clinical outcome, which is largely recapitulated in an independent UTUC cohort. Sequencing of urine sediment-derived DNA has a high diagnostic value for UTUC with 82.2% sensitivity and 100% specificity. These results provide a solid basis for better diagnosis and management of UTUC.

Activation of EHF via STAT3 phosphorylation by LMP2A in Epstein-Barr virus-positive gastric cancer.

Epstein-Barr virus (EBV) is associated with approximately 10% of gastric cancers (GCs). We previously showed that EBV infection of gastric epithelial cells induces aberrant DNA methylation in promoter regions, which causes silencing of critical tumor suppressor genes. Here, we analyzed gene expressions and active histone modifications (H3K4me3, H3K4me1, and H3K27ac) genome-widely in EBV-positive GC cell lines and in vitro EBV-infected GC cell lines to elucidate the transcription factors contributing to tumorigenesis through enhancer activation. Genes associated with "signaling of WNT in cancer" were significantly enriched in EBV-positive GC, showing increased active ß-catenin staining. Genes neighboring activated enhancers were significantly upregulated, and EHF motif was significantly enriched in these active enhancers. Higher expression of EHF in clinical EBV-positive GC compared with normal tissue and EBV-negative GC was confirmed by RNA-seq using The Cancer Genome Atlas cohort, and by immunostaining using our cohort. EHF knockdown markedly inhibited cell proliferation. Moreover, there was significant enrichment of critical cancer pathway-related genes (eg, FZD5) in the downstream of EHF. EBV protein LMP2A caused upregulation of EHF via phosphorylation of STAT3. STAT3 knockdown was shown to inhibit cellular growth of EBV-positive GC cells, and the inhibition was rescued by EHF overexpression. Our data highlighted the important role of EBV infection in gastric tumorigenesis via enhancer activation.

Helicobacter pylori CagA elicits BRCAness to induce genome instability that may underlie bacterial gastric carcinogenesis.

Infection with CagA-producing Helicobacter pylori plays a causative role in the development of gastric cancer. Upon delivery into gastric epithelial cells, CagA deregulates prooncogenic phosphatase SHP2 while inhibiting polarity-regulating kinase PAR1b through complex formation. Here, we show that CagA/PAR1b interaction subverts nuclear translocation of BRCA1 by inhibiting PAR1b-mediated BRCA1 phosphorylation. It hereby induces BRCAness that promotes DNA double-strand breaks (DSBs) while disabling error-free homologous recombination-mediated DNA repair. The CagA/PAR1b interaction also stimulates Hippo signaling that circumvents apoptosis of DNA-damaged cells, giving cells time to repair DSBs through error-prone mechanisms. The DSB-activated p53-p21Cip1 axis inhibits proliferation of CagA-delivered cells, but the inhibition can be overcome by p53 inactivation. Indeed, sequential pulses of CagA in TP53-mutant cells drove somatic mutation with BRCAness-associated genetic signatures. Expansion of CagA-delivered cells with BRCAness-mediated genome instability, from which CagA-independent cancer-predisposing cells arise, provides a plausible "hit-and-run mechanism" of H. pylori CagA for gastric carcinogenesis.

Risk for lymph node metastasis in Epstein-Barr virus-associated gastric carcinoma with submucosal invasion.

OBJECTIVES: Epstein-Barr virus-associated gastric cancer (EBVGC) has been reported to be associated with a low risk for lymph node metastasis (LNM). However, the curative criteria for endoscopic submucosal dissection (ESD) for submucosal EBVGC (pT1b-EBVGC) remain unclear. Our study aimed to investigate the risk factors for LNM in pT1b-EBVGC. METHODS: This was a retrospective multicenter study at five institutes in Japan. We reviewed medical records and extracted all pT1b-EBVGC cases that met the following criteria: (i) histologically proven submucosal gastric cancer; (ii) surgical or endoscopic resection between January 2000 and December 2016; and (iii) presence of Epstein-Barr virus (EBV) in tumor cells verified by EBV-encoded small RNA in situ hybridization (EBER-ISH). The association between clinicopathological factors and LNM were assessed using multivariable logistic regression analysis. RESULTS: A total of 185 pT1b-EBVGC cases were included in the analysis. LNM was found in nine cases (4.9%). Multivariable logistic regression analysis demonstrated that lymphatic invasion (OR 9.1; 95% CI 2.1-46.1) and submucosal invasion ≥4000 µm (OR 9.2; 95% CI 1.3-110.3) were significant risk factors for LNM. When we focused on pT1b-EBVGC without lymphatic invasion and with submucosal invasion <2000 µm, the rate of LNM was 0% (0/96, 95% CI 0-3.8%). CONCLUSIONS: Our findings indicated that lymphatic invasion and submucosal invasion ≥4000 µm were significant risk factors for LNM. ESD could be an appropriate option for pT1b-EBVGC without lymphatic invasion and with submucosal invasion <2000 µm.

CD70 in Thymic Squamous Cell Carcinoma: Potential Diagnostic Markers and Immunotherapeutic Targets.

CD70 - a ligand protein of CD27 on lymphocytes - is expressed in a large spectrum of malignancies. It is an attractive target for antibody-based therapy and several clinical trials are currently being conducted. However, there is no evidence regarding the expression of CD70 and its relationship with expression of programmed death ligand-1 (PD-L1) and CD27+ tumor-infiltrating lymphocytes (TIL) in formalin-fixed paraffin-embedded (FFPE) tissues of thymic tumors. FFPE tissues of thymic squamous cell carcinoma (TSCC) (operative specimens, n = 31; biopsy specimens, n = 11), thymoma (n = 60), thymic carcinoid (n = 3), and lung squamous cell carcinoma (LSCC) (n = 30) were analyzed immunohistochemically. Immunoreactivity for CD70 was semi-quantitatively scored according to the proportion of positive tumor cells. Moreover, the densities of CD27-positive intratumoral TIL (iTIL) and stromal TIL of TSCC were assessed and survival was compared. Most TSCC cases (87%; 27/31) were CD70-positive. In contrast, all thymoma and thymic carcinoid cases were CD70-negative. In LSCC cases, CD70-positivity was significantly lower than TSCC cases (20%; 6/30). Biopsy and resected specimens obtained from the same patients demonstrated a consistent staining pattern (6/6 patients). The proportion of CD70-positive TSCC was comparable with those of CD5 (87%) and CD117 (90%). Correlation between CD70 and PD-L1 expression score was observed. There was no significant difference in survival between the CD70-high and CD70-low expression groups. Meanwhile, patients with CD27-positive iTIL-high tumors exhibited better survival than those with iTIL-low tumors. This tendency was weaker in the CD70-high subset. CD70 immunohistochemistry is useful in diagnosing TSCC. CD70 may prevent anti-tumor immunity via CD27. Immunotherapy targeting the CD70-CD27 axis may be a promising option for the treatment of TSCC.

Comprehensive immunohistochemical analysis of the gastrointestinal and Müllerian phenotypes of 139 ovarian mucinous cystadenomas.

Mucinous cystadenoma is one of the most common benign ovarian neoplasms. The immunophenotypes and histogenetic relationships of mucinous cystadenomas with a Müllerian-type epithelium have not been fully explored. We elucidated the direction of differentiation of the mucinous epithelium that constitutes mucinous cystadenomas. Special attention was paid to the existence of gastrointestinal (GI)-type mucinous epithelium, and its association with background Müllerian-type epithelium. Immunohistochemistry was performed in 139 cases of mucinous cystadenoma to evaluate the expression of Claudin-18 (CLDN18), a novel marker of gastric differentiation; CDX2, a marker of intestinal differentiation; and estrogen receptor (ER), a marker of Müllerian differentiation. We found that GI differentiation characterized by CLDN18 and/or CDX2 positivity was observed in mucinous epithelium of most mucinous cystadenomas (129/139 cases, 93%). In a subset of these cases, the tumor was composed of mucinous epithelium exhibiting an intermediate GI and Müllerian phenotype (CLDN18+/CDX2±/ER+). Of note, in 12 cases, a transition from background Müllerian-type epithelium to mucinous epithelium with GI differentiation was identified. A minor subset (6%) of mucinous cystadenomas was considered a pure Müllerian type because the epithelium exhibited a CLDN18-/CDX2-/ER + immunophenotype. In conclusion, mucinous cystadenomas consist of three major subtypes: GI, Müllerian, and intermediate types. Most mucinous cystadenomas are GI-type, and they should be considered a precursor of GI-type mucinous borderline tumors. The existence of intermediate-type mucinous cystadenomas, and areas of transition from Müllerian-type to GI-type epithelium suggest that GI-type mucinous epithelium can arise from Müllerian duct derivatives or surface epithelium exhibiting Müllerian metaplasia in the ovary.