[Synchronous gastrointestinal stromal tumors of the rectum and duodenum: a case report].

An 85-year-old woman with anemia underwent colonoscopy, which revealed a 25-mm submucosal tumor with ulceration in the lower rectum. Histological examination of a biopsy showed that the lesion was a gastrointestinal stromal tumor (GIST). Subsequent esophagogastroduodenoscopy revealed a submucosal tumor in the duodenum, and examination of a biopsy obtained by endoscopic ultrasound-guided fine-needle aspiration also confirmed that this lesion was a GIST. The rectal lesion was surgically resected to control bleeding and was confirmed as a GIST histologically. Simultaneous development of GISTs in the rectum and duodenum is extremely rare.

[Three cases of pedunculated gastric hamartomatous inverted polyps resected endoscopically].

We report three cases of pedunculated gastric hamartomatous inverted polyps (HIPs) that were successfully treated by endoscopic polypectomy. The first case involved an 87-year-old woman with mild anemia. Esophagogastroduodenoscopy (EGD) revealed a pedunculated, reddish polyp located at the greater curvature of the upper stomach. The second case involved a 34-year-old woman in whom a pedunculated polyp was found at the gastric fundus during routine EGD. The third patient was a 59-year-old woman with epigastric discomfort. EGD revealed a pedunculated polyp in the gastric fundus. Polypectomy was successfully performed in all three cases. Histological examination revealed that the tumors comprised submucosal proliferation of cystically dilated gastric glands and hyperplastic glands;thus, we diagnosed gastric HIPs, which are rare and typically difficult to diagnose. Gastric HIPs should be considered in the differential diagnosis of elevated gastric lesions.

Cancer-associated fibroblasts might sustain the stemness of scirrhous gastric cancer cells via transforming growth factor-ß signaling.

Cancer-associated fibroblasts (CAFs) have recently been implicated in tumor growth and metastasis in gastric cancer. Cancer stem cells (CSCs) have been proposed to have an important role in cancer progression. The aim of this study was to clarify the effect of CAFs on CSCs characteristics in gastric carcinoma. Scirrhous gastric cancer cell lines, OCUM-12 and OCUM-2MD3, and non-scirrhous gastric cancer cell lines, MKN-45 and MKN-74, were used. OCUM-12/side population (SP) cells and OCUM-2MD3/SP cells were sorted by flow cytometry as CSC-rich cells from the parent cells. CaF-37 was established from the tumoral gastric specimens as CAFs. Flow cytometric analysis of SP fraction, spheroid colony assay, and RT-PCR analysis of CSC markers were performed to identify CSCs properties. Effect of CAFs on the tumorigenicity by OCUM-12/SP cells was examined using nude mice. CAF CM significantly increased the percentages of the SP fraction of OCUM-12/SP and OCUM-2MD3/SP cells, but not that of MKN-45/SP and MKN-74/SP cells. Taken together, CM from CaF-37 significantly increased the number of spheroid colonies and the expression level of CSC markers of OCUM-12/SP and OCUM-2MD3/SP cells. These stimulating-activities by CM were significantly decreased by TGFß inhibitors, but not FGFR and cMet inhibitor. Tumorigenicity by subcutaneous coinoculation of OCUM-12/SP cells with CAFs was significantly high in comparison with that by OCUM-12/SP cells alone. Phospho-Smad2 expression level was significantly increased by co-inoculation with CAFs. These findings suggested that CAFs might regulate the stemness of CSCs in scirrhous gastric cancer by TGFß signaling.

Cancer-associated orthotopic myofibroblasts stimulates the motility of gastric carcinoma cells.

Tumor progression has been recognized as the product of evolving crosstalk between cancer cells and the surrounding stromal cells. Cancer-associated orthotopic myofibroblasts may be linked to the progression of gastric carcinomas. To understand the significance of orthotopic myofibroblasts, we examined the effects of cancer-associated orthotopic myofibroblasts on the malignant phenotype of gastric cancer cells. Three human gastric cancer cell lines (OCUM-2MD3, OCUM-12, MKN-45) and four human gastric fibroblast cell lines (cancer-associated orthotopic fibroblast [CaF]-29, CaF-33, normal orthotopic fibroblast [NF]-29, NF-33) were used. The cancer-associated orthotopic fibroblast cell lines CaF-29 and CaF-33 were established from a tumoral gastric wall, and normal orthotopic fibroblast NF-29 and NF-33 were established from a non-tumoral gastric wall. Fibroblasts that were α-smooth muscle actin-positive were defined as myofibroblasts. We examined the effects of cancer-associated orthotopic myofibroblasts on the aggressiveness of gastric cancer cells by wound-healing assay, invasion assay, and RT-PCR. The ratios of myofibroblasts in CaF-29 (33%) and CaF-33 (46%) were significantly (P < 0.001) greater than those in NF-29 (11%) or NF-33 (13%). Although all four orthotopic fibroblast lines increased the motility of gastric cancer cells, including migration and invasion ability, the motility-stimulating activity of cancer-associated fibroblasts (CaF-29 and CaF-33) was significantly higher than that of normal fibroblasts (NF-29 and NF-33). These motility-stimulating activities of cancer-associated orthotopic fibroblasts were downregulated by Smad2 siRNA treatment and anti-transforming growth factor-ß neutralizing antibody. These findings suggest that cancer-associated orthotopic myofibroblasts may play an important role in the progression of gastric cancers and that transforming growth factor-ß produced by myofibroblasts may be one of the factors associated with the aggressiveness of gastric carcinoma cells.

Role of the stemness factors sox2, oct3/4, and nanog in gastric carcinoma.

BACKGROUND: Cancer stem cells have been proposed to be responsible for cancer tumorigenicity, and then to persist in tumors as a distinct population and cause relapse and metastasis. Recently, the stemness factors Sox2, Oct3/4, and Nanog were associated with induced pluripotent stem cells, suggesting a correlation between these stemness factors and cancer stem cells. We therefore investigated the role of stemness factors in the tumorigenesis of human gastric cancer. MATERIALS AND METHODS: A total of 290 patients who had undergone resection of a primary gastric cancer at our institute were enrolled. A curative R0 resection was performed for 253 of 290 patients, and the remaining 37 patients were treated with a palliative resection. The expression levels of Sox2, Oct3/4, and Nanog were analyzed by immunohistochemistry. RESULTS: Sox2, Oct3/4, and Nanog expression were positive in 159 (55%), 129 (44%), and 28 (10%) of 290 gastric cancers, respectively. There was a statistically significant correlation between Sox2-positive or Oct3/4-negative expression and invasion depth, lymph node metastasis, or lymphatic invasion. In 253 patients with a curative resection, the prognosis of patients with Sox2-positive tumors or Oct3/4-negative tumors was significantly (P < 0.01 or P = 0.04, log-rank) worse than that of patients with Sox2-negative or Oct3/4-positive tumors, respectively. A multivariate analysis revealed the expression of Sox2 or Oct3/4 to be an independent prognostic factor (P = 0.01 or P = 0.04). CONCLUSIONS: Sox2-positive expression or Oct3/4-negative expression might be associated with invasion of gastric cancer. Sox2 and Oct3/4 might be independent prognostic factors for patients with gastric cancer.

[Lumpectomy and sentinel lymph node navigation surgery for breast cancer under local anesthesia].

We studied and analyzed therapeutic outcomes of a radical surgery under local anesthesia for breast cancer in our department. Subjects were 42 patients with breast cancer whose diagnoses were definitely made before surgery. Indications were: localized DCIS diagnosed preoperatively; invasive carcinoma less than 3 cm in tumor diameter on ultrasound; and clinically tumors with negative axillary lymph nodes. Operative procedures included lumpectomy associated with sentinel lymph node navigation biopsy. We could perform the operation under local anesthesia in all of the 42 patients, and were not demanded to shift from local to general anesthesia. Two patients had sentinel lymph nodes metastasis. Surgical stumps were positive in 14 patients( 33.3%). None of serious complications were encountered. Today's radical operation under local anesthesia for breast cancer is a useful procedure as minimally invasive surgery as for the indications employed in this study.

Easy clinical predictor for low BCAA to tyrosine ratio in chronic liver disease patients with hepatocellular carcinoma: Usefulness of ALBI score as nutritional prognostic marker.

BACKGROUND/AIM: Low branched-chain amino acid (BCAA) to tyrosine ratio (BTR) is known as an indicator of amino acid imbalance. We elucidated usefulness of newly developed albumin-bilirubin (ALBI) score as alternative methods of BTR in patients with naïve hepatocellular carcinoma (HCC) retrospectively. MATERIALS/METHODS: In 842 patients with HCC and without BCAA supplementation (71 years, male 614, Child-Pugh A:B:C = 689:116:37), relationships among BTR and clinical features were evaluated. Of those, 438 patients, with Milan criteria HCC, treated curatively were divided into the high-BTR (>4.4) (n = 293) and low-BTR (≤4.4) (n = 145) groups. The prognostic value of BTR was evaluated using inverse probability weighting (IPW) with propensity score. RESULTS: The low-BTR group showed worse prognosis than the other (3-, 5-, 10-year overall survival rates: 88.9% vs. 86.3%/70.5% vs. 78.1%/38.1% vs. 52.3%, respectively; p < 0.001). Multivariate Cox-hazard analysis adjusted for IPW showed elderly (≥65 years) HR 2.314, p = 0.001), female gender (HR 0.422, p < 0.001), ECOG PS ≥2 (HR 3.032, p = 0.002), low platelet count (HR 1.757, p = 0.010), and low BTR (≤4.4) (HR 1.852, p = 0.005) to be significant prognostic factors. Both serum albumin level (r = 0.370, p < 0.001) and ALBI score (r = -0.389, p < 0.001) showed a significant relationship with BTR. Child-Pugh class B, modified ALBI grade (mALBI) 2a, and mALBI 2b predictive values for BTR were 3.589, 4.509, and 4.155 (AUC range: 0.735-0.770), respectively, while the predictive value of ALBI score for low-BTR (≤4.4) was -2.588 (AUC 0.790). CONCLUSION: ALBI score -2.588 was a predictor for low-BTR (≤4.4), which was prognostic factors for early HCC patients, and at least patients with mALBI 2b might have an amino acid imbalance.

Factors Related to Sleeping Disorder Due to Pruritus in Patients with Chronic Liver Disease.

Objective This study evaluated cases of pruritus, which is known to be associated with sleep disorder, in chronic liver disease (CLD) patients. Methods Questionnaires were given to 339 enrolled CLD outpatients in winter (November 2019 to March 2020) and again in summer (April to October 2020) (median interval: 104 days). Relative changes in symptoms shown by a visual analogue scale (VAS) and Kawashima's pruritus score between winter and summer were evaluated in Study 1 (n=199), while Study 2 examined the clinical features of patients with sleep disorder based on the results of the second questionnaire (n=235, median age 70 years old; 141 men, liver cirrhosis 37%). Results Study 1. There was a significant relationship in VAS between daytime and nighttime for each season, as well as between winter and summer for each time period (p<0.001). A comparison of Kawashima's pruritus scores for the daytime and nighttime showed no significant seasonal differences (p=0.436 and 0.828, respectively). When Kawashima's score increased, so did the average VAS for both daytime (0:1:2:3:4=0.4±0.2:1.4±0.9:3.0±1.8:5.9±2.1:6.2±2.3) and nighttime (0:1:2:3:4=0.3±0.1:1.4±1.5:3.5±2.3:6.7±2.6:6.9±1.8) (p<0.001 for both). Study 2. Twenty subjects (8.5%) complained of sleep disorder. An elevated FIB-4 index (≥3.07) showed a good predictive value for sleep disorder (p<0.01). The cut-off for the daytime and nighttime VAS values for existing sleep disorder were 1.6 [area under the curve (AUC) 0.901] and 3.4 (AUC 0.931). The respective sensitivity, specificity, and positive and negative predictive values for sleep disorder based on Kawashima's score (≥2) were 0.85, 0.28, 0.10, and 0.95 for the daytime and 1.00, 0.29, 0.12, and 1.00 for the nighttime. Conclusion Intervention against pruritus is recommended in CLD patients with a high Kawashima's score (≥2) in any season, especially with an elevated FIB-4 index.

Expression of a hypoxia-associated protein, carbonic anhydrase-9, correlates with malignant phenotypes of gastric carcinoma.

BACKGROUND/AIMS: Gastric cancers are characterized by a heterogeneously hypoxic environment. Hypoxia might stimulate the malignant potential of cancer cells. The purpose of our study was to clarify the significance of hypoxia in gastric carcinoma by evaluating the expression of a hypoxic marker, namely carbonic anhydrase-9 (CA-9). METHODS: A total of 265 patients who had undergone a resection of the primary tumor and were confirmed histologically to have sporadic gastric cancer were enrolled in this study. We evaluated the immunohistochemical expression of CA-9 in the paraffin-embedded specimens of 265 gastric adenocarcinomas. RESULTS: The CA-9 expression was positive in 88 (33%) of 265 gastric carcinomas. The CA-9 expression level was significantly high in cases of type 4 carcinoma (60%, p < 0.001) and diffuse type carcinoma (41%, p < 0.001), and significantly correlated with the invasion depth (p < 0.001), lymph node metastasis (p < 0.001) and lymphatic invasion p = 0.002). The prognosis for CA-9-positive patients was significantly poorer than that of CA-9-negative patients (p = 0.003, log-rank). CONCLUSION: Hypoxia might be associated with aggressive tumor phenotypes of gastric carcinomas. The hypoxic marker CA-9 may be a useful prognostic indicator.

Expression and regulation of the immediate-early gene product Arc in the accessory olfactory bulb after mating in male rat.

Recent studies of the accessory olfactory bulb have shown that the expression of immediate-early genes, e.g., c-fos, c-jun and egr-1, can be used as a marker of neuronal activity in response to pheromonal cues. In this study, we analyzed the expression pattern, in response to mating, of the novel immediate-early gene product Arc (an activity-regulated cytoskeleton-associated protein). Arc is hypothesized to play a role in activity-dependent neuronal plasticity in the hippocampus. In a control group of male rats, only a small number of Arc-immunoreactive cells were observed in the accessory olfactory bulb. In a mating group, however, a marked increase in the number of Arc-immunoreactive cells was observed only in the granule cell layer of the accessory olfactory bulb. The increase in the number of Arc-immunoreactive cells after mating was similar to that observed for other immediate-early genes. However, for the mating group, the increase in Arc-positive cells was limited to the granule cell layer. Granule cells have been shown to exhibit a strong synaptic plasticity in response to pheromonal stimulation. From these findings we suggest that Arc plays an important role in neuronal plasticity in the accessory olfactory bulb.

A comparative immunocytochemical study of development and regeneration of chemosensory neurons in the rat vomeronasal system.

Vomeronasal neurons undergo continuous neurogenesis during development and after neuronal injury. We used immunocytochemical methods to compare different stages of the vomeronasal organ development to those of regeneration following vomeronasal nerve transection. At E15 and at 6 to 10 days after injury, nestin-positive cells were observed throughout the sensory epithelium. We did not find nestin immunoreactivity to be localized to the boundary region of the epithelium. The early appearance and wide distribution of nestin-positive cells suggests that they represent chemosensory precursor cells that develop and migrate vertically in the epithelium. Vomeronasal receptor cells degenerated 6 to 8 days after nerve transection, but axon terminals in the accessory olfactory bulb (AOB) continued to show the presence of the chemosensory specific marker (OMP) for up to ten days, a significant finding observed in this study. It is likely that the distance from the site of nerve transection may contribute to differences in the time course of anterograde and retrograde axon degradation. OMP-positive neurons were observed in the normal adult epithelium and to a much lesser extent 10-60 days after recovery from nerve transection. Axons from regenerated receptor cells did not reach the AOB during this time period. This failure to reestablish connections with target cells in the AOB could explain why OMP-positive cells were rarely observed among the regenerated cells in the vomeronasal epithelium.

Rapid induction of Arc is observed in the granule cell dendrites in the accessory olfactory bulb after mating.

The activity-regulated cytoskeleton-associated protein (Arc), encoded by the immediate early gene arc, is enriched in the brain and is hypothesized to play a role in the activity-dependent neuronal plasticity in the hippocampus. In the present study, the time course of Arc expression during the post-mating period was determined immunocytochemically, and the localization of Arc in the neurons in the accessory olfactory bulb (AOB) of female mice after mating was analyzed using immunocytochemical electron microscopy. Transient increases in the number of Arc-immunoreactive cells were observed in the glomerular, mitral/tufted cell and granule cell layers of the AOB after mating. In particular, the increase in the granule cell layer was remarkable, and larger than the increases in the other layers. In addition, electron microscopic observation revealed that Arc immunoreactivity was in the dendrites of the granule cells 1.5 h after mating. These results indicate that expression of Arc protein is induced rapidly and transiently in granule cell dendrites after mating. It is postulated that Arc protein has a role in the neuronal plasticity of the AOB after mating.

Mating behavior induces differential Arc expression in the main and accessory olfactory bulbs of adult rats.

The expression of activity-related cytoskeleton-associated protein, Arc, could be useful as a marker for neuronal activity. We investigated Arc-immunoreactivity in both the accessory olfactory bulb (AOB) and the main olfactory bulb (MOB) of adult male rats in response to mating or exposure to female pheromones. Mating behavior strongly enhanced the Arc-immunoreactivity in the granule cell layer of the AOB. However, the enhancement of Arc-immunoreactivity by mating behavior was not observed in the MOB. These results showed that Arc-immunoreactivity was enhanced when the AOB received both afferent and efferent information during mating behavior. Hence, the expression of Arc in the AOB directly associates the pheromonal information with mating behavior. The AOB will provide a useful model to investigate the function of Arc protein.

Hypoxia stimulates the EMT of gastric cancer cells through autocrine TGFß signaling.

Epithelial mesenchymal transition (EMT) is considered to be correlated with malignancy of cancer cells and responsible for cancer invasion and metastasis. We previously reported that distant metastasis was associated with hypoxia in gastric cancer. We therefore investigated the effect of hypoxic condition on EMT of gastric cancer cells. Gastric cancer cells were cultured in normoxia (21% O2) or hypoxia (1% O2) for 24 h. EMT was evaluated as the percentage of spindle-shaped cells in total cells. Effect of transforming growth factor ß1 (TGFß1) or tyrosine kinase inhibitors on the EMT was evaluated. The expression level of TGFß1 and TGFßR was evaluated by real time RT-PCR. The TGFß1 production from cancer cells was measured by ELISA. Hypoxia stimulated EMT of OCUM-2MD3 and OCUM-12 cells, but not that of OCUM-2M cells. The expression level of TGFß1 mRNA under hypoxia was significantly higher than that under normoxia in all of three cell lines. The expression level of TGFßR mRNA was significantly increased by hypoxia in OCUM-2MD3 cells, but not in OCUM-2M cells. TGFßR inhibitor, SB431542 or Ki26894, significantly suppressed EMT of OCUM-2MD3 and OCUM-12. TGFß1 production from OCUM-2MD3 and OCUM-12 cells was significantly increased under hypoxia in comparison with that under normoxia. These findings might suggest that hypoxia stimulates the EMT of gastric cancer cells via autocrine TGFß/TGFßR signaling.

Effects of acute and chronic hypoxia on the radiosensitivity of gastric and esophageal cancer cells.

AIM: The aim of this study was to examine the effects of hypoxia on radiosensitivity and to analyze the mechanisms responsible for radiation resistance in gastric and esophageal cancer. MATERIALS AND METHODS: A gastric cancer cell line, OCUM-12, and an esophageal cancer cell line, TE-6, were used. The effects of hypoxia with irradiation on the growth-activity, cell cycle distribution, and gene expression were examined. RESULTS: Both acute and chronic hypoxia decreased radiosensitivity of cancer cells. The radiosensitivity of chronic hypoxic cells was significantly enhanced by reoxygenation. Acute and chronic all hypoxia reduced the percentage of cells in the G(2)/M and S phases, respectively. In acute hypoxia, the mRNA expression of BRCA1 and BRCA2 was reduced in cancer cells. Reoxygenation increased the expression of BRCA1 and BRCA2. CONCLUSION: Hypoxia is associated with radiation resistance. Therefore, reoxygenation may enhance the radiosensitivity of hypoxic cells. BRCA1 and BRCA2 may be associated with factors for radiation resistance by regulation of cell cycle progression.

Myofibroblasts are associated with the progression of scirrhous gastric carcinoma.

Fibroblasts, particularly myofibroblasts, affect the malignant progression of cancer cells in vitro. However, to date few reports have addressed the clinical significance of myofibroblasts in the gastric cancer microenvironment. This study examined the correlation between myofibroblast expression and clinicopathological features in gastric carcinoma. A total of 265 primary gastric tumors resected by gastrectomy were stained with antibodies against α-smooth muscle actin and vimentin. Stromal cells positive for vimentin were considered to be fibroblasts. Myofibroblasts were defined as fibroblasts positive for α-smooth muscle staining. Myofibroblast-positive gastric carcinoma was established when myofibroblasts accounted for more than 25% of fibroblasts in the cancer stroma. Myofibroblast expression was positive in 92 (35%) of the 265 gastric carcinomas. Myofibroblast expression showed a significantly (p<0.001) high frequency in advanced gastric cancers (76 of 146), in comparison to the early stage cancers (16 of 119). Taken together, there was a statistically significant correlation between myofibroblast expression and scirrhous type gastric cancer (p<0.001), lymph node metastasis (p<0.001), lymphatic invasion (p<0.001) and peritoneal dissemination (p=0.005). The prognosis of patients with tumors positive for myofibroblast expression was significantly (p<0.001) worse, while a multivariate analysis revealed that myofibroblast expression was not an independent prognostic factor. These findings suggest that myofibroblasts are associated with scirrhous gastric cancer. Overexpression of myofibroblasts may therefore be a useful prognostic indicator of gastric carcinoma.