Modular Design Platform for Activatable Fluorescence Probes Targeting Carboxypeptidases Based on ProTide Chemistry.

Carboxypeptidases (CPs) are a family of hydrolases that cleave one or more amino acids from the C-terminal of peptides or proteins and play indispensable roles in various physiological and pathological processes. However, only a few highly activatable fluorescence probes for CPs have been reported, and there is a need for a flexibly tunable molecular design platform to afford a range of fluorescence probes for CPs for biological and medical research. Here, we focused on the unique activation mechanism of ProTide-based prodrugs and established a modular design platform for CP-targeting florescence probes based on ProTide chemistry. In this design, probe properties such as fluorescence emission wavelength, reactivity/stability, and target CP can be readily tuned and optimized by changing the four probe modules: the fluorophore, the substituent on the phosphorus atom, the linker amino acid at the P1 position, and the substrate amino acid at the P1' position. In particular, switching the linker amino acid at position P1 enabled us to precisely optimize the reactivity for target CPs. As a proof-of-concept, we constructed probes for carboxypeptidase M (CPM) and prostate-specific membrane antigen (also known as glutamate carboxypeptidase II). The developed probes were applicable for the imaging of CP activities in live cells and in clinical specimens from patients. This design strategy should be useful in studying CP-related biological and pathological phenomena.

Prevalence and diagnostic significance of non-invasive follicular thyroid neoplasm with papillary-like nuclear features in Japan-A multi-institutional study.

This multi-institutional study investigated non-invasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) frequency and its diagnostic significance in Japan. We reviewed 4008 thyroid nodules resected in six institutions before NIFTP was proposed. Overall, 26 cases diagnosed as non-invasive encapsulated follicular variant of papillary thyroid carcinoma (PTC) and 145 cases of follicular thyroid adenoma (FTA) were included. Of these nodules, 80.8% and 31.0%, respectively, were NIFTPs. In five institutions, NIFTPs were more commonly found in FTA than in PTC nodules. When NIFTP was included with PTC, the overall prevalence was 2.3%, with rates in five institutions below 5.0% (0.8%-4.4%). One NIFTP case with nuclear score 3 revealed nodal metastasis 2.5 years post-resection, and the carcinoma cells were immunohistochemically positive for BRAF. FTAs or NIFTPs with nuclear score 2 did not metastasize. NIFTP was more common among FTA than among PTC nodules, possibly due to underdiagnosis of PTC on nuclear findings. Considering the clinical findings, molecular pathogenesis, and therapeutic strategy in Japan, NIFTP with nuclear score 2 is not different from FTA, and use of this entity terminology is not meaningful. In contrast, NIFTP with nuclear score 3 has potential for metastasis and BRAFV600E mutation. Therefore, in NIFTP cases, nuclear scores 2 and 3 should be separately reported.

Low-carbohydrate diets adversely impact the skin of a mouse model of photoaging exposed to ultraviolet B radiation.

The study results regarding the effects of low-carbohydrate (LC) diets remain controversial; hence further research is required to assess their safety. Here, we examined whether LC diets cause skin damage in C57BL/6J mice. Six-week-old female mice (n = 20) were fed an LC (protein/fat/carbohydrate energy ratio = 35:45:20) or control diet ad libitum for eight weeks, after which their backs were shaved, and a subset of the mice were exposed to ultraviolet B radiation thrice per week. Ultraviolet B irradiation induced wrinkle formation on the skin surface, and thickening of the epidermis, which was also noticeable in the LC diet-fed mice in the absence of ultraviolet B radiation. Meanwhile, the number of epidermal melanocytes and degree of horny layer keratosis increased in the LC diet-fed mice following ultraviolet B irradia-tion. mRNA expression analysis of the liver and skin showed decreased levels of the antioxidant enzyme superoxide dismutase 1 following ultraviolet B irradiation only in the LC diet-fed mice. Alternatively, the expression of pro-inflammatory cytokines, tumor necrosis factor-α and interleukin-1ß, increased in response to ultraviolet B radiation and LC diet intake. Hence, LC diets may adversely affect skin morphology and exacerbate the effects of ultraviolet B irradiation, which may be associated with anti-oxidant dysfunction.

Stromal tiny black dots, like "sugar-coated", of von Kossa stain is a diagnostic clue to hyalinizing trabecular tumor of the thyroid gland.

Hyalinizing trabecular tumor (HTT) is a rare low-grade tumor, and a prominent feature is the basement membranous stroma. We assume that such characteristic stromal findings of HTT are related to calcium deposition, and examined HTT samples by von Kossa special staining. There has been no report describing von Kossa special staining for such stroma. We collected 12 cases of HTT and 30 cases of papillary thyroid carcinoma (PTC) that had matched age, gender, tumor size, and surgical procedure characteristics as a control group. We compared the staining pattern and degree of von Kossa positivity between HTT and PTC, and a grading system of von Kossa stain was adopted to highlight differences between them. On von Kossa staining, all HTT revealed many tiny black dots around vessels in the hyalinized stroma, like "sugar-coated", and a high degree of calcium deposition in most cases, whereas PTC showed sparse stromal calcification in some cases. The degree of von Kossa staining was significantly different between the two groups. This is the first report describing abundant tiny black dots, like a "sugar-coated" appearance, of von Kossa stain in HTT. Here, we propose this finding can be a useful diagnostic clue to HTT.

Molecular alterations of coexisting thyroid papillary carcinoma and anaplastic carcinoma: identification of TERT mutation as an independent risk factor for transformation.

Thyroid papillary carcinoma is the most common endocrine neoplasm and generally carries a favorable prognosis. However, a small subset of papillary carcinomas transforms into anaplastic carcinoma, an undifferentiated cancer with a dismal prognosis. Recent studies using next-generation sequencing revealed the genomic landscape of papillary carcinoma and anaplastic carcinoma. However, risk factors for anaplastic transformation in papillary carcinoma remain obscure. In the present study, we investigated molecular alterations of papillary carcinoma and anaplastic carcinoma components in 27 tumors in which anaplastic carcinoma coexisted with antecedent papillary carcinoma. We conducted direct sequencing for BRAF, TERT promoter and PIK3CA, and immunohistochemistry for p53, TTF-1 and subunits of the SWI/SNF complex (ARID1A, ARID1B, ATRX, SMARCA2, SMARCA4, SMARCB1, and PBRM1). BRAFV600E and TERT promoter mutated at the rate of 90% and 95%, respectively, and these mutational statuses were almost identical between the papillary carcinoma and anaplastic carcinoma components. PIK3CA mutation was positive in 33% of our samples with a heterogeneous mutation pattern of the papillary carcinoma and anaplastic carcinoma components. Aberrant expression of p53 and loss of TTF-1 were present in 63 and 59%, respectively, and these two alterations were confined to the anaplastic carcinoma components. There was a loss of the SWI/SNF complex in a subset of the tumors with a heterogeneous pattern of the papillary carcinoma and anaplastic carcinoma components: SMARCA4 in 4% and PBRM1 in 4%. In a multivariate comparison between the antecedent papillary carcinoma components and control papillary carcinomas without anaplastic transformation, TERT promoter mutation was independently associated with anaplastic transformation. Collectively, papillary carcinoma-derived anaplastic carcinomas are characterized by BRAF and TERT promoter mutations, and these mutations occur prior to anaplastic transformation. Alterations of PIK3CA and the SWI/SNF complex are relatively rare and temporally heterogeneous. Of note, a papillary carcinoma harboring TERT promoter mutation is at higher risk for anaplastic transformation.

Development of an Activatable Fluorescent Probe for Prostate Cancer Imaging.

Technology to visualize small prostate cancers is urgently needed because of the difficulty of discriminating prostate cancer from normal tissue with the naked eye, and a fluorescence imaging method would be advantageous. Here, we describe the design and synthesis of a fluorogenic probe (Ac-KQLR-HMRG) that is activated by hepsin and matriptase (proteases over-expressed in prostate cancer). Ac-KQLR-HMRG exhibited significant turn-on fluorogenicity in the presence of hepsin (180-fold) and matriptase (80-fold) and allowed specific fluorescence imaging of various prostate cancer cell line in vitro. In addition, the probe enabled rapid imaging (within 1-10 min) of small prostate cancer nodules in mouse models of disseminated peritoneal tumor and orthotopic tumor.

Rapid imaging of lung cancer using a red fluorescent probe to detect dipeptidyl peptidase 4 and puromycin-sensitive aminopeptidase activities.

Rapid identification of lung-cancer micro-lesions is becoming increasingly important to improve the outcome of surgery by accurately defining the tumor/normal tissue margins and detecting tiny tumors, especially for patients with low lung function and early-stage cancer. The purpose of this study is to select and validate the best red fluorescent probe for rapid diagnosis of lung cancer by screening a library of 400 red fluorescent probes based on 2-methyl silicon rhodamine (2MeSiR) as the fluorescent scaffold, as well as to identify the target enzymes that activate the selected probe, and to confirm their expression in cancer cells. The selected probe, glutamine-alanine-2-methyl silicon rhodamine (QA-2MeSiR), showed 96.3% sensitivity and 85.2% specificity for visualization of lung cancer in surgically resected specimens within 10 min. In order to further reduce the background fluorescence while retaining the same side-chain structure, we modified QA-2MeSiR to obtain glutamine-alanine-2-methoxy silicon rhodamine (QA-2OMeSiR). This probe rapidly visualized even borderline lesions. Dipeptidyl peptidase 4 and puromycin-sensitive aminopeptidase were identified as enzymes mediating the cleavage and consequent fluorescence activation of QA-2OMeSiR, and it was confirmed that both enzymes are expressed in lung cancer. QA-2OMeSiR is a promising candidate for clinical application.

Development of a fluorescent probe library enabling efficient screening of tumour-imaging probes based on discovery of biomarker enzymatic activities.

Fluorescent probes that can selectively detect tumour lesions have great potential for fluorescence imaging-guided surgery. Here, we established a library-based approach for efficient screening of probes for tumour-selective imaging based on discovery of biomarker enzymes. We constructed a combinatorial fluorescent probe library for aminopeptidases and proteases, which is composed of 380 probes with various substrate moieties. Using this probe library, we performed lysate-based in vitro screening and/or direct imaging-based ex vivo screening of freshly resected clinical specimens from lung or gastric cancer patients, and found promising probes for tumour-selective visualization. Further, we identified two target enzymes as novel biomarker enzymes for discriminating between tumour and non-tumour tissues. This library-based approach is expected to be an efficient tool to develop tumour-imaging probes and to discover new biomarker enzyme activities for various tumours and other diseases.

Rapid and Accurate Visualization of Breast Tumors with a Fluorescent Probe Targeting α-Mannosidase 2C1.

Accurate detection of breast tumors and discrimination of tumor from normal tissues during breast-conserving surgery are essential to reduce the risk of misdiagnosis or recurrence. However, existing probes show substantial background signals in normal breast tissues. In this study, we focus on glycosidase activities in breast tumors. We synthesized a series of 12 fluorescent probes and performed imaging-based evaluation on surgically resected human breast specimens. Among them, the α-mannosidase-reactive fluorescent probe HMRef-αMan detected breast cancer with 90% sensitivity and 100% specificity. We identified α-mannosidase 2C1 as the target enzyme and confirmed its overexpression in various breast tumors. We found that fibroadenoma, the most common benign breast lesion in young woman, tends to have higher α-mannosidase 2C1 activity than malignant cancer. Combined application of green-emitting HMRef-αMan and a red-emitting γ-glutamyltranspeptidase probe enabled efficient dual-color, dual-target optical discrimination of malignant and benign tumors.

Epstein-Barr virus-associated gastric carcinoma: a distinct carcinoma of gastric phenotype by claudin expression profiling.

Epstein-Barr virus (EBV)-associated gastric carcinoma (GC) is a distinct subtype with characteristic clinicopathological features. To better characterize its cellular characteristics, 43 cases of EBV-associated GC, 68 cases of EBV-negative GC, and non-neoplastic gastric mucosa in adults and fetuses were examined immunohistochemically. We quantified the expression of the major tight-junction protein claudin (CLDN) -1, -3, -4, -7, and -18 together with gastric mucins (MUC5AC and MUC6), intestinal mucin (MUC2), and CD10. EBV-associated GC showed a high frequency of CLDN18 expression (84%) and a low frequency of CLDN3 expression (5%). This expression profile corresponded to that of normal gastric epithelium in adults and fetuses. Almost half of the EBV-associated GC cases demonstrated gastric mucin expression, whereas the other half lacked mucin or CD10 expression. In contrast, as demonstrated by the expression profiles of CLDN3 and CLDN18, EBV-negative GC comprised a heterogeneous group of four different CLDN phenotypes: gastric, intestinal, mixed, and an undifferentiated type with variable expression patterns of mucins. These results indicate that EBV-associated GC is considerably homogenous with regard to cellular differentiation and that it preserves well the nature of the cells of origin. EBV-associated GC may undergo distinct carcinogenic processes, which differ from those of EBV-negative GC.

Low-Carbohydrate Diet Inhibits Different Advanced Glycation End Products in Kidney Depending on Lipid Composition but Causes Adverse Morphological Changes in a Non-Obese Model Mice.

Low carbohydrate diets (LC diets) have been noted for adverse health effects. In addition, the effect of lipid composition on an LC diet is unclear. In this study, we used an LC diet containing two different lipids, lard (LC group) and medium-chain triglyceride oil (MCT-LC group), to examine the effect of an LC diet in non-obese mice. Male C57BL/6J mice were fed the control diet or one of the experimental diets ad libitum for 13 weeks. Increased renal weight and glomerular hypertrophy, as well as enlargement of intraglomerular small vessels with wall thickening, were seen in the LC and MCT-LC groups. Renal AMP-activated protein kinase activity was significantly decreased only in the LC diet group. On the other hand, epididymal adipose tissue weight and adipocyte area were markedly decreased only in the MCT-LC group. A positive effect was also observed in the kidney, where different advanced glycation end products, Nε-(carboxyethyl)-lysine and Nε-(carboxymethyl)-lysine, were inhibited depending on the lipid composition of the LC diet. Our findings suggest that, in non-obese conditions, low dietary intake of carbohydrates had both positive and negative impacts. The safety of diets low in carbohydrates, including the effects of fatty acid composition, requires further investigation.

Epstein-Barr virus and gastric carcinoma: virus-host interactions leading to carcinoma.

Epstein-Barr virus (EBV)-associated gastric carcinoma (GC) is a distinct subgroup of GC, comprising 10% of all cases of GC. EBV-associated carcinoma is the monoclonal growth of EBV-infected epithelial cells, and it represents a model of virus-host interactions leading to carcinoma. EBV-infected cells express several latent proteins (latency I program of viral latent gene expression) in EBV-associated GC. However, latent membrane protein 2A (LMP2A) up-regulates the cellular survivin gene through the NFkB pathway, conferring resistance to apoptotic stimuli on the neoplastic cells. EBV-associated GC also shows characteristic abnormality, that is, global and non-random CpG island methylation of the promoter region of many cancer-related genes. Since the viral genes are also regulated by promoter methylation in the infected cells, the DNA methylation mechanism specific to EBV-associated GC may be an exaggeration of the cellular mechanism, which is primarily for defense against foreign DNA. Production of several immunomodulator molecules, inducing tumor-infiltrating lymphocyte and macrophages, serves to form the characteristic histologic pattern in EBV-associated GC. The proposed sequence of events within the mucosa is as follows: EBV infection of certain gastric stem cells; expression of viral latent genes; abnormality of signal pathways caused by viral gene products; DNA methylation-mediated repression of tumor suppressor genes; and monoclonal growth of EBV-infected cells through interaction with other etiologic factors. Potentially useful therapeutic approaches to EBV-associated GC are those that utilize the virus-host interactions, such as bortezomib-induced and viral enzyme-targeted radiotherapy.

Rapid detection of papillary thyroid carcinoma by fluorescence imaging using a γ-glutamyltranspeptidase-specific probe: a pilot study.

BACKGROUND: Nodular lesions of the thyroid gland, including papillary thyroid carcinoma (PTC), may be difficult to diagnose by imaging, such as in ultrasonic echo testing, or by needle biopsy. Definitive diagnosis is made by pathological examination but takes several days. A more rapid and simple method to clarify whether thyroid nodular lesions are benign or malignant is needed. Fluorescence imaging with γ-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG) uses γ-glutamyltranspeptidase (GGT), a cell-surface enzyme, to hydrolyze the γ-glutamyl peptide and transfer the γ-glutamyl group. GGT is overexpressed in several cancers, such as breast, lung, and liver cancers. This imaging method is rapid and useful for detecting such cancers. In this study, we tried to develop a rapid fluorescence detection method for clinical samples of thyroid cancer, especially papillary carcinoma. METHODS: Fluorescence imaging with gGlu-HMRG was performed to detect PTC using 23 surgically resected clinical samples. A portable imaging device conveniently captured white-light images and fluorescence images with blue excitation light. Hematoxylin-eosin (HE) staining was used to evaluate which fluorescent regions coincided with cancer, and immunohistochemical examination was used to detect GGT expression. RESULTS: All 16 PTC samples exhibited fluorescence after topical application of gGlu-HMRG, whereas the normal sections of each sample showed no fluorescence. HE staining revealed that each fluorescent region corresponded to a region with carcinoma. The PTC samples also exhibited GGT expression, as confirmed by immunohistochemistry. CONCLUSIONS: All PTC samples were detected by fluorescence imaging with gGlu-HMRG. Thus, fluorescence imaging with gGlu-HMRG is a rapid, simple, and powerful detection tool for PTC.

CpG island methylation status in gastric carcinoma with and without infection of Epstein-Barr virus.

PURPOSE: EBV-associated gastric carcinoma shows global CpG island methylation of the promoter region of various cancer-related genes. To further clarify the significance of CpG island methylator phenotype (CIMP) status in gastric carcinoma, we investigated methylation profile and clinicopathologic features including overall survival in four subgroups defined by EBV infection and CIMP status: EBV-associated gastric carcinoma and EBV-negative/CIMP-high (H), EBV-intermediate (I), and EBV-negative (N) gastric carcinoma. EXPERIMENTAL DESIGN: Methylation-specific PCR was applied to 106 gastric carcinoma cases. CIMP-N, CIMP-I, and CIMP-H status was determined by the number (0, 1-3, and 4-5, respectively) of methylated marker genes (LOX, HRASLS, FLNc, HAND1, and TM), that were newly identified as highly methylated in gastric cancer cell lines. The methylation status of 10 other cancer-related genes (p14, p15, p16, p73, TIMP-3, E-cadherin, DAPK, GSTP1, hMLH1, and MGMT) was also evaluated. RESULTS: Nearly all (14 of 15) of EBV-associated gastric carcinoma exhibited CIMP-H, constituting a homogenous group (14%). EBV-negative gastric carcinoma consisted of CIMP-H (24%), CIMP-I (38%), and CIMP-N (24%). EBV-associated gastric carcinoma showed significantly higher frequencies of methylation of cancer-related genes (mean number +/- SD = 6.9 +/- 1.5) even if compared with EBV-negative/CIMP-H gastric carcinoma (3.5 +/- 1.8). Among EBV-negative gastric carcinoma subgroups, CIMP-H gastric carcinoma showed comparatively higher frequency of methylation than CIMP-I or CIMP-N, especially of p16 and hMLH1. CIMP-N gastric carcinoma predominantly consisted of advanced carcinoma with significantly higher frequency of lymph node metastasis. The prognosis of the patients of CIMP-N was significantly worse compared with other groups overall by univariate analysis (P = 0.0313). CONCLUSION: The methylation profile of five representative genes is useful to stratify gastric carcinomas into biologically different subgroups. EBV-associated gastric carcinoma showed global CpG island methylation, comprising a pathogenetically distinct subgroup in CIMP-H gastric carcinoma.

Brain edema with clasmatodendrosis complicating ataxia telangiectasia.

Ataxia-telangiectasia is a chronic progressive disorder affecting the nervous and immune systems, caused by a genetic defect in the ATM protein. Clasmatodendrosis, a distinct form of astroglial death, has rarely been reported in ataxia-telangiectasia. Neuropathology of our patient disclosed diffuse edema of the cerebral and cerebellar white matter with prominent clasmatodendrosis, implicating ATM in the regulation of astroglial cell death.

Phenotype analysis by MUC2, MUC5AC, MUC6, and CD10 expression in Epstein-Barr virus-associated gastric carcinoma.

BACKGROUND: Gastric marker mucins (MUC5AC and MUC6) and intestinal marker molecules (MUC2 and CD10) have been used to determine the cell lineage of epithelial cell of gastric carcinoma (GC). METHODS: To clarify the characteristics of Epstein-Barr virus (EBV)-associated GC, 18 cases were immunohistochemically evaluated along with 56 cases of EBV-negative GC. RESULTS: MUC2 expression was lower in EBV-associated GC: immunostaining grades 0, 1, 2, 3, and 4 were observed in 10, 6, 1, 1, and 0 cases of EBV-associated GC, respectively, and in 18, 11, 15, 6, and 6 cases of EBV-negative GC, respectively (P = 0.013). CD10 positivity (grades 2-4) in EBV-associated GC was 6%, significantly lower than in EBV-negative GC (34%) (P = 0.030). When phenotypes of GC were categorized by the combined positivities of gastric markers (either MUC5AC or MUC6) and intestinal markers (either MUC2 or CD10), EBV-associated GC included primarily null (44%) and gastric (39%) types, but EBV-negative GC comprised null (7%), gastric (30%), intestinal (27%), and mixed (36%) types. The age of patients with gastric types was significantly younger for both EBV-associated GC and EBV-negative GC cases. CONCLUSIONS: Neoplastic epithelial cells of EBV-associated GC did not express MUC2 or CD10, and most of them were categorized as null or gastric types. EBV infection may occur in the epithelial cells of null or gastric phenotypes, which may be devoid of transdifferentiation potential toward intestinal phenotypes.

Platelet-derived growth factor-A and vascular endothelial growth factor-C contribute to the development of pulmonary tumor thrombotic microangiopathy in gastric cancer.

Pulmonary tumor thrombotic microangiopathy is a rare but lethal complication in cancer-bearing patients, particularly those with gastric cancer. It is characterized by cancer cell emboli with marked intimal proliferation. In the present study, we tried to elucidate the pathogenesis of pulmonary tumor thrombotic microangiopathy, notably angiogenic factors specific for cancer cells lodged in pulmonary arteries. An autopsy series of gastric cancer (51 cases) was reviewed for pulmonary tumor thrombotic microangiopathy and pulmonary tumor cell emboli without intimal proliferation. Pathological and immunohistochemical characteristics were compared between two groups. In eight cases in muscular pulmonary arteries, tumor thrombotic microangiopathy was noted, and in three cases pulmonary tumor emboli without intimal proliferation was noted. Histological features of pulmonary tumor thrombotic microangiopathy included small nests or single cancer cells accompanied by intimal proliferation, whereas in pulmonary tumor emboli large cell nests prevailed. By immunohistochemistry, in pulmonary tumor thrombotic microangiopathy, cancer cells expressed platelet-derived growth factor-A (7/8 cases) and vascular endothelial growth factor-C (8/8) more frequently than in pulmonary tumor emboli (0/3 and 1/3; P = 0.02 and P = 0.055, respectively). Expression of tissue factor, vascular endothelial growth factor-A and -D, osteopontin, fibroblast growth factor-2, and platelet-derived growth factor-B was similar in both groups. Platelet-derived growth factor-A and vascular endothelial growth factor-C might induce intimal proliferation in pulmonary arteries and contribute to the development of pulmonary tumor thrombotic microangiopathy.

Enrichment of stem-like cell population comprises transformation ability of Epstein-Barr virus latent membrane protein 2A for non-transformed cells.

Epstein-Barr virus (EBV) is a representative human oncogenic virus that causes malignancies of various cell lineages. LMP2A, an EBV-encoded latent membrane protein, is expressed in EBV-associated malignancies of various cell lineages. LMP2A caused visible tumor formation transplanted in nude mice when transferred to immortalized non-transformed fibroblasts, NIH3T3. LMP2A-expressing cells showed higher ability of colony formation in soft agar than empty vector-transfected control cells, although the expression of LMP2A did not cause focus transformation in low serum concentrations. LMP2A expression increased the size of Hoechst 33,342 dye excreting side population (SP), in which cancer-initiating cells or cancer stem-like cells were enriched. SP increase by LMP2A was also responsible for colony formation in soft agar. The LMP2A-mediated SP increase depended on the activations of Stat3, MEK/ERK, and PI3K pathways, and on upregulation of HMGA2. Enrichment of SP, stem-like cells, by LMP2A promoted the transformation capability of LMP2A from non-transformed cells. The enrichment of stem-like cell population by a virus-encoded factor might explain the oncogenic functions of oncogenic viruses.

SOX9 expression and its methylation status in gastric cancer.

SOX9 is a member of the SOX [Sry-related high-mobility group (HMG) box] family and is required for the development and differentiation of multiple cell lineages. To clarify the significance of SOX9 in gastric carcinoma (GC), immunohistochemical expression of SOX9 and the CpG island methylation status of SOX9 were evaluated and compared with clinicopathological factors including overall survival. SOX9 expression was immunohistochemically evaluated in 382 GC tumors and the methylation status was examined in 121 GC tumors. SOX9 expression and its methylation status in six GC cell lines, their Epstein-Barr virus (EBV)-infected cell lines, and two EBV-associated GC cell lines was also examined. The SOX9 expression increased from non-neoplastic mucosa to early cancer. High expression of SOX9 was seen in 212 cases (56%). SOX9 expression was inversely related to advanced tumor stage, vessel infiltration, nodal metastasis, and EBV infection. Fifty-eight (48%) of 121 GC tumors had a methylated promoter in GC and the methylated status was related to low expression. The expression and methylation status were not related to prognosis. Three of six cell lines had increased methylation through EBV infection and decreased SOX9 expression. Upregulation of SOX9 is related to GC development. Downregulation by promoter methylation is related to GC progression and EBV infection. SOX9 is closely related to GC carcinogenesis and EBV-associated GC carcinogenesis.

ARID1A expression loss in gastric cancer: pathway-dependent roles with and without Epstein-Barr virus infection and microsatellite instability.

The AT-rich interactive domain 1A gene (ARID1A), which encodes one of the subunits in the Switch/Sucrose Nonfermentable chromatin remodeling complex, carries mutations and is responsible for loss of protein expression in gastric carcinoma, particularly with Epstein-Barr virus (EBV) infection and a microsatellite instability-high phenotype. We used immunohistochemistry to investigate the significance of ARID1A loss in 857 gastric carcinoma cases, including 67 EBV(+) and 136 MLH1-lost gastric carcinomas (corresponding to a microsatellite instability-high phenotype). Loss of ARID1A expression was significantly more frequent in EBV(+) (23/67; 34 %) and MLH1-lost (40/136; 29 %) gastric carcinomas than in EBV(-)MLH1-preserved (32/657; 5 %) gastric carcinomas (P < 0.01). Loss of ARID1A correlated with larger tumor size, advanced invasion depth, lymph node metastasis, and poor prognosis in EBV(-)MLH1-preserved gastric carcinoma. A correlation was found only with tumor size and diffuse-type histology in MLH1-lost gastric carcinoma, but no correlation was observed in EBV(+) gastric carcinoma. Loss of ARID1A expression in EBV(+) gastric carcinoma was highly frequent in the early stage of gastric carcinoma, although EBV infection did not cause downregulation of ARID1A: EBV-positive nasopharyngeal carcinomas (n = 8) and lymphomas (n = 15) failed to show loss of ARID1A, and EBV infection did not cause loss of ARID1A in gastric carcinoma cell lines. Taken together, loss of ARID1A may be an early change in carcinogenesis and may precede EBV infection in gastric epithelial cells, while loss of ARID1A promotes cancer progression in gastric cancer cells without EBV infection or loss of MLH1 expression. Loss of ARID1A has different and pathway-dependent roles in gastric carcinoma.