Sun-Hwi Hwang

Cancer spheres from gastric cancer patients provide an ideal model system for cancer stem cell research

Cancer stem cells have been hypothesized to drive the growth and metastasis of tumors. Because they need to be targeted for cancer treatment, they have been isolated from many solid cancers. However, cancer stem cells from primary human gastric cancer tissues have not been isolated as yet. For the isolation, we used two cell surface markers: the epithelial cell adhesion molecule (EpCAM) and CD44. When analyzed by flow cytometry, the EpCAM+/CD44+ population accounts for 4.5% of tumor cells. EpCAM+/CD44+ gastric cancer cells formed tumors in immunocompromised mice; however, EpCAM−/CD44−, EpCAM+/CD44− and EpCAM−/CD44+ cells failed to do so. Xenografts of EpCAM+/CD44+ gastric cancer cells maintained a differentiated phenotype and reproduced the morphological and phenotypical heterogeneity of the original gastric tumor tissues. The tumorigenic subpopulation was serially passaged for several generations without significant phenotypic alterations. Moreover, EpCAM+/CD44+, but not EpCAM−/CD44−, EpCAM+/CD44− or EpCAM−/CD44+ cells grew exponentially in vitro as cancer spheres in serum-free medium, maintaining the tumorigenicity. Interestingly, a single cancer stem cell generated a cancer sphere that contained various differentiated cells, supporting multi-potency and self-renewal of a cancer stem cell. EpCAM+/CD44+ cells had greater resistance to anti-cancer drugs than other subpopulation cells. The above in vivo and in vitro results suggest that cancer stem cells, which are enriched in the EpCAM+/CD44+ subpopulation of gastric cancer cells, provide an ideal model system for cancer stem cell research.

Overexpression of NRG1 promotes progression of gastric cancer by regulating the self-renewal of cancer stem cells

BackgroundGastric cancer stem cells (GCSCs) have been successfully isolated from patients. However, the molecular mechanisms underlying the self-renewal of GCSCs and their relationship with the microenvironment are poorly characterized.MethodsGCSCs and cancer-associated fibroblasts (CAFs) were cultured directly from gastric cancer patients. The self-renewal of GCSCs was assayed by sphere formation assay and in vivo tumorigenicity. Expression of neuregulin1 (NRG1) was examined by immunohistochemistry, real-time PCR and western blotting.ResultsCAFs increased the self-renewal of GCSCs by secreting NRG1. NRG1 activated NF-κB signaling and this activation regulated GCSC self-renewal. Moreover, NF-κB-active GCSCs were tumorigenic, however NF-κB-inactive GCSCs were not. The overexpression of NRG1 in stromal cells and cancer cells was observed in the tumor tissues of gastric cancer patients and was associated with clinical stage lymph node metastasis and survival in gastric cancer patients. In addition, we also found that NRG1 can regulate the proliferation and invasion of gastric cancer cells.ConclusionsThese results indicate that NRG1, which can be secreted by CAFs or cancer cells, promotes progression of gastric cancer by regulating the self-renewal of GCSCs and its overexpression is associated with a prognosis of gastric cancer.

LAP2 Is Widely Overexpressed in Diverse Digestive Tract Cancers and Regulates Motility of Cancer Cells

Background Lamina-associated polypeptides 2 (LAP2) is a nuclear protein that connects the nuclear lamina with chromatin. Although its critical roles in genetic disorders and hematopoietic malignancies have been described, its expression and roles in digestive tract cancers have been poorly characterized. Methods To examine the expression of LAP2 in patient tissues, we performed immunohistochemistry and real-time PCR. To examine motility of cancer cells, we employed Boyden chamber, wound healing and Matrigel invasion assays. To reveal its roles in metastasis in vivo, we used a liver metastasis xenograft model. To investigate the underlying mechanism, a cDNA microarray was conducted. Results Immunohistochemistry in patient tissues showed widespread expression of LAP2 in diverse digestive tract cancers including stomach, pancreas, liver, and bile duct cancers. Real-time PCR confirmed that LAP2β is over-expressed in gastric cancer tissues. Knockdown of LAP2β did not affect proliferation of most digestive tract cancer cells except pancreatic cancer cells. However, knockdown of LAP2β decreased motility of all tested cancer cells. Moreover, overexpression of LAP2β increased motility of gastric and pancreatic cancer cells. In the liver metastasis xenograft model, LAP2β increased metastatic efficacy of gastric cancer cells and mortality in tested mice. cDNA microarrays showed the possibility that myristoylated alanine-rich C kinase substrate (MARCKS) and interleukin6 (IL6) may mediate LAP2β-regulated motility of cancer cells. Conclusions From the above results, we conclude that LAP2 is widely overexpressed in diverse digestive tract cancers and LAP2β regulates motility of cancer cells and suggest that LAP2β may have utility for diagnostics and therapeutics in digestive tract cancers.

Local tissue reaction after injection of contrast media on gastric wall of mouse: experimental study for application of contrast media to computed tomography lymphography

Purpose Computed tomography (CT) lymphography is a simple technique of sentinel node navigation but tissue reaction after injection of contrast media has not been reported yet. Methods Ninety mice used in this study were divided into three groups: lipiodol, iopamidol, and normal saline. The test compounds were given by submucosal injection to the gastric wall of anesthetized mice. The specimens were subjected to histopathological examination. Results The mean grades of acute inflammatory response after iopamidol and lipiodol injection were significantly higher than control group. However, there was no significant difference between iopamidol and lipiodol injection. The mean grade of chronic inflammatory response and fibrosis showed no differences between groups. The presence or absence of fibrinoid necrosis and mesothelial hyperplasia showed no statistical differences at each time point between groups. The foam cell, which is similar to human signet ring cell carcinoma, were not identified in normal saline and iopamidol group, but were detected by postoperative day 7 in lipiodol group. Conclusion We conclude that iopamidol and lipiodol when used as a contrast media of CT lymphography is an available material for preoperative sentinel node navigation surgery for gastric cancer with an acceptable incidence of pathological alterations in a mouse model. Our results are potentially useful to clinical (human) application.

Differential expression of a WD protein during squamous differentiation of tracheal epithelial cells

The lining of the trachea consists of a pseudostratified, mucociliary epithelium that under a variety of conditions, such as vitamin A deficiency, toxic and mechanical injury, becomes a stratified squamous epithelium. Several in vitro cell culture models have been established to study the process of differentiation of airway epithelium. Such studies have indicated that mucosecretory differentiation of tracheal epithelial cells can be modulated by substratum. This study was undertaken to understand molecular mechanisms of squamous differentiation in tracheal epithelia. Primary cultured tracheal cells grown on uncoated filters were differentiated to single layer of squamous cells, whereas cells were grown as stratified columanar cells on collagen‐I coated filters. The responses to secretagogues were altered according to culture conditions. DD‐PCR revealed that FAK and a WD protein expression was increased in squamous tracheal epithelia. Expression of a WD protein was changed by the treatment of retinoic acid in various epithelial cells. These results indicated that squamous differentiation of tracheal cells changes the expression of a variety of genes, and that the experimental model for this study can be employed to study molecular mechanisms of squamous differentiation in airway epithelial cells. J. Cell. Biochem. 86: 194–201, 2002.

Atraumatic Liver Retraction Using Nelaton Catheters During Totally Laparoscopic Gastrectomy

This study introduces a novel technique for liver retraction during laparoscopic gastrectomy and assesses its impact on postoperative recovery. This study included 139 patients in whom Nelaton catheters (n=57) or Nathanson retractors (n=82) were used for liver retraction. Serum liver enzyme levels were measured preoperatively and on the first, second, third, fifth, and seventh postoperative days. Clinicopathologic features and postoperative recovery variables between the 2 groups were compared. The aspartate aminotransferase, alanine aminotransferase, and C-reactive protein levels were significantly lower (P<0.001, P<0.001, and P=0.007, respectively), and the day of first flatus, the day of initiating a soft diet, and the length of hospital stay were shorter in the Nelaton catheter U-shaped retractor group than those seen in the Nathanson retractor group (P=0.035, P=0.002, and P=0.024, respectively). Atraumatic liver retraction with Nelaton catheters is recommended in laparoscopic gastrectomy.

Epidemiologic Study of Human Epidermal Growth Factor Receptor 2 Expression in Advanced/Metastatic Gastric Cancer: an Assessment of Human Epidermal Growth Factor Receptor 2 Status in Tumor Tissue Samples of Gastric and Gastro-Esophageal Junction Cancer

Purpose The Trastuzumab for gastric cancer (GC) trial identified human epidermal growth factor receptor 2 (HER2) as a predictor of successful treatment with trastuzumab (HER2 receptor targeting agent) among patients with advanced/metastatic GC. To date, the prevalence of HER2 overexpression in the Korean population is unknown. The present study aimed to assess the incidence of HER2 positivity among GC and gastroesophageal (GE) junction cancer samples and the relationship between HER2 overexpression and clinicopathological characteristics in Korean patients. Materials and Methods Tumor samples collected from 1,695 patients with histologically proven GC or GE junction enrolled at 14 different hospitals in Korea were examined. After gathering clinicopathological data of all patients, HER2 status was assessed by immunohistochemistry (IHC) at each hospital, and IHC 2+ cases were subjected to silver-enhanced in situ hybridization at 3 central laboratories. Results A total of 182 specimens tested positive for HER2, whereas 1,505 tested negative. Therefore, the overall HER2-positive rate in this study was 10.8% (95% confidence interval=9.3%–12.3%). The HER2-positive rate was higher among intestinal-type cases (17.6%) than among other types, and was higher among patients older than 70 years and 50 years of age, compared to other age groups. Conclusions Our evaluation of the HER2 positivity rate (10.8%) among Korean patients with GC and GE junction indicated the necessity of epidemiological data when conducting studies related to HER2 expression in GC and GE junction.

Risk factors for lymph node metastasis in mucosal gastric cancer and re-evaluation of endoscopic submucosal dissection

Purpose The selection of the appropriate treatment strategy for patients with mucosal gastric cancer (MGC) remains controversial. In the present study, we aimed to determine the risk factors for lymph node (LN) metastasis in MGC and reassess the role of endoscopic submucosal dissection (ESD). Methods We examined 1,191 MGC patients who underwent curative gastrectomy between January 2005 and December 2014. We determined the clinicopathologic risk factors for LN metastasis among the MGC patients. Results Among 1,191 patients with MGC, 42 patients (3.5%) had LN metastasis. Univariate analysis indicated that age ≤ 50 years (P = 0.045), tumor invasion to the muscularis mucosa (P < 0.001), tumor size > 2 cm (P = 0.014), presence of ulceration (P = 0.01), diffuse type as per Lauren classification (P = 0.005), and undifferentiated-type histology (P = 0.001) were associated with LN metastasis. Moreover, multivariate analysis indicated that tumor invasion to the muscularis mucosa (P = 0.001; odds ratio [OR], 4.909), presence of ulceration (P = 0.036; OR, 1.982), and undifferentiated-type histology (P = 0.025; OR, 4.233) were independent risk factors for LN metastasis. In particular, LN metastasis was observed in some MGC cases with indications for ESD, including absolute indications (1 of 179, 0.6%) and expanded indications (9 of 493, 1.8%). Conclusion Although MGC patients can be treated via ESD, we recommend that they undergo a more aggressive treatment strategy if they have tumor invasion to the muscularis mucosa, ulceration, or undifferentiated-type histology in the final pathology report.

Mixed adenoneuroendocrine carcinoma in the stomach: a case report with a literature review

Mixed adenoneuroendocrine carcinoma (MANEC) is a rare disease that was first defined in the 2010 World Health Organization classification of endocrine tumors. We present the case of a 65-year-old man with an ulcerative depressed lesion measuring 3 cm in diameter and found in the lower gastric body. It was diagnosed as a MANEC, and we performed subtotal gastrectomy with D2 lymph node dissection. The patient is still alive, and no recurrence was observed at a 12-month follow-up. Most MANECs tend to have a poor prognosis. Curative resection, including an adequate lymph node dissection, should be considered, and intense follow-up is needed.

Effect of extramucin pools in gastric cancer patients

Purpose Mucinous gastric adenocarcinoma (MGC) is defined by the World Health Organization as a gastric adenocarcinoma with >50% extracellular mucin pools within the tumors. In this study, we attempted to analyze the clinicopathologic features of patients pathologically diagnosed as gastric cancer with lower than 50% tumor volume of extracellular mucin pool adenocarcinoma (LEMPC). We compared MGC versus nonmucinous gastric adenocarcinoma (NMGC). We were used in abbreviations LEMPC for NMGC including extracellular mucin pool. Methods Files of 995 patients with gastric cancer NMGC (n = 935), MGC (n = 20), LEMPC (n = 40) who underwent curative resection at Pusan National University Yangsan Hospital from December 2008 to December 2013 were retrospectively analyzed. All pathologic reports after curative resection and evaluated clinicopathologic features were reviewed to identify the effect of extracellular mucin pools in gastric cancer. Results Compared with the NMGC patients, the clinicopathological features of MGC patients were as follows: more frequent open surgery, larger tumor size, more advanced T stage and N stage, more positive lymph node metastasis, and perineural invasion. LEMPC patients showed similar features compared with NMGC patients. MGC and LEMPC patients showed similar clinicopathological features, except T stage and lymph node metastasis. Conclusion LEMPC can be thought of as a previous step of MGC. It is reasonable to consider LEMPC patients in the diagnostic criteria of MGC, and to adequately treat.