Increased Cellular Levels of MicroRNA-9 and MicroRNA-221 Correlate with Cancer Stemness and Predict Poor Outcome in Human Breast Cancer.

Background /Aims: Recent studies of microRNA (miRNA) involvement in tumorigenesis have indicated the critical role of these non-coding small RNAs in malignant transformation, but the prognostic role, if any, of miRNAs in breast cancer remains undetermined. Therefore, we assessed the prognostic significance of microRNA-9 (miR-9) and miR-221 in breast cancer toward the goal of understanding the contribution(s) of these miRNAs to cancer cell stemness. METHODS: The level of each of miR-9 and miR-221 in 206 paired laser capture microdissected tumor cells and non-tumor cells was determined by quantitative reverse transcription-PCR (qRT-PCR). The relationship between the miRNA signature and clinicopathological data and prognosis of breast cancer was assessed. Identification of a stem cell-enriched side population was achieved with fluorescence-activated cell sorting and a sphere-forming assay. Wound healing, Boyden chamber assays, and western blotting were used to study the contribution of each miRNA to tumor cell migration and invasion. RESULTS: We found that induction of miR-9 and miR-221 mimics conferred side-population cells to form spheroidal tumor colonies in suspension culture that maintained the mesenchymal stem-cell potential in non-invasive MCF-7 breast cancer cells. In contrast, knockdown of both miR-9 and miR-221 in invasive MDA-MB-231 breast cancer cells dramatically decreased the number of side-population colonies with stem cell-like potency, which reduced the capacity for tumor-cell renewal, invasion, and migration. Clinically, the mean proportion of miR-9- or miR-221-overexpressing cells was significantly greater in tumor cells compared with non-tumor cells (P < 0.05). Increased levels of miR-9 and miR-221 in breast tissue portended a significantly elevated risk of progression to malignancy with respect to larger tumor size, poor differentiation, late-stage evolution, lymph-node metastasis (P < 0.05), and lower overall survival (Ptrend = 0.017; eight-year follow-up). CONCLUSION: Our findings provide strong evidence that miR-9 and miR-221 can enhance the generation of cancer stem cells to yield an invasive phenotype and that overexpression of these miRNAs predicts a poor outcome for breast cancer patients.

The effects of perceived stress and life style leading to breast cancer.

Researchers conducted a study in a Taiwanese medical center from June 2009 to June 2011 to investigate the relations of perceived stress and lifestyle to breast cancer. A total of 157 cases and 314 controls completed a structured questionnaire. Using multiple logistic regression models, high perceived stress (adjusted odds ratio [AOR] = 1.65; 95% confidence interval [CI], 1.10-2.47), less than 1,000 kcal of physical activity expenditure per week (AOR, 2.17; 95% CI, 1.39-3.39), and high intake of fried and stir-fried food (AOR, 1.86; 95% CI, 1.24-2.77) were positively associated with breast cancer. Breast cancer was related to joint interactions between high perceived stress and alcohol intake of 11.0 g or more per day (AOR, 2.91; 95% CI, 1.23-6.86), smoking at least one cigarette per day (AOR, 2.52; 95% CI, 1.16-5.47), intake of less than 100 ml of green tea per day (AOR, 2.47; 95% CI, 1.40-4.38), physical activity of less than 1,000 kcal per week (AOR, 3.36; 95% CI, 1.77-6.36), high fried and stir-fried food intake (AOR, 3.18; 95% CI, 1.79-5.63), and high meat and seafood intake (AOR, 1.89; 95% CI, 1.09-3.27). Perceived stress, when combined with potentially risky lifestyle behaviors, may be a contributing factor to breast cancer.

Left Nonrecurrent Laryngeal Nerve with Situs Inversus Totalis.

The recurrent laryngeal nerve (RLN), a branch of the vagus nerve, supplies the motor and sensation function of the larynx. Generally, RLN detours around the right subclavian artery on the right side and the aortic arch on the left side. In a rare anatomical variant, called nonrecurrent laryngeal nerve (NRLN), the nerve takes an aberrant path rather than descending into the thorax as usual. First reported in 1823, NRLN is a rare anomaly arising almost exclusively on the right side, reported in 0.3-0.8% of people, and associated with vascular anomalies of embryonic aortic arch development. The atypical vascular pattern of aberrant subclavian artery (arteria lusoria) running behind the trachea and esophagus allows the vagus nerve to pass freely, which then directly branches out as NRLN at the level of the larynx. On the other hand, cases of left NRLN, only reported in 0.004% of people, are all accompanied by significant pathologies such as situs inversus totalis with opposite vascular pattern of left aberrant subclavian artery. This rare anatomical variation is clinically important, as NLRN is a major risk factor for iatrogenic injury during thyroidectomy, parathyroidectomy, and other invasive procedures in the head and neck region.

Overcoming the difficulties in laparoscopic management of contracted gallbladders with gallstones: possible role of fundus-down approach.

BACKGROUND: The aim of this study is to compare efficacy and complications between fundus-down and conventional laparoscopic cholecystectomy (LC) in treating contracted gallbladders with gallstones. METHODS: Between January 1999 and May 2008, 64 patients with contracted gallbladders and gallstones were included in the study. Main outcome measures included conversion rate, complication rate, bile duct injury rate, operation time, and postoperative stay. RESULTS: The average postoperative hospital stay for fundus-down technique was 5 ± 3 days, and 7 ± 3 days for conventional technique (P = 0.003). The conversion rate and complication rate were 0% (0/33) and 3.00% (1/33) for fundus-down technique, and 32.3% (10/31) and 22.6% (7/31) for conventional technique (P = 0.0009 and 0.02, respectively). In subgroup analysis, fundus-down LC seemed to lower the bile duct injury rate from 2/31 (6.5%) to 0/33 (0%) compared with 6/1,468 (0.4%) (P = 0.01 between 6.5% and 0.4% vs. P = 1.00 between 0% and 0.4%). CONCLUSIONS: It appears that fundus-down laparoscopic cholecystectomy is associated with lower conversion and complication rates and shorter postoperative hospital stay as compared with conventional laparoscopic cholecystectomy when used to treat patients with contracted gallbladders and gallstones.

True cytokeratin 8/18 immunohistochemistry is of no use in distinguishing between primary endocervical and endometrial adenocarcinomas in a tissue microarray study.

The choice of appropriate therapeutic plans for primary endocervical adenocarcinomas and endometrial adenocarcinomas depends on the site of origin of the tumor. The purpose of this study was to make clear whether the immunohistochemistry of the true cytokeratin 8/18 monoclonal antibody (Leica Microsystems, Newcastle, United Kingdom), instead of CAM 5.2 (Becton Dickinson Biosciences, San Jose, CA), has potential use in distinguishing between endocervical adenocarcinomas and endometrial adenocarcinomas. A tissue microarray was constructed using paraffin-embedded, formalin-fixed tissues from 34 hysterectomy specimens, including 14 endocervical adenocarcinomas and 20 endometrial adenocarcinomas. Using the Bond-Max autostainer (Leica Microsystems) and the associated Bond Refine Polymer Detection Kit, tissue array sections were immunostained with cytokeratin 8, 18, and 8/18 commercially available antibodies. The immunohistochemical expressions of all 3 markers, cytokeratin 8, 18, and 8/18 showed nonsignificant (P>0.05) frequency differences between the immunostaining results (positive vs. negative) in tumors of both gynecologic adenocarcinomas. Although CAM 5.2 has been reported to be helpful in distinguishing between primary endocervical adenocarcinomas and endometrial adenocarcinomas, we could not verify this point of view using the true cytokeratin 8/18 monoclonal antibody (Leica Microsystems). It has often been mistakenly cited that CAM 5.2 reacts with cytokeratin 8 and 18, and the results herein confer that there is a wrong impression that cytokeratin 8/18 is differentially expressed in these 2 gynecologic malignancies. In conclusion, the true cytokeratin 8/18 monoclonal antibody is of no use in distinguishing between primary endocervical adenocarcinomas and endometrial adenocarcinomas.

Laparoscopic primary closure of common bile duct combined with percutaneous cholangiographic drainage for treating choledocholithiasis.

The aim of this study was to compare the efficacy and safety of laparoscopic primary closure of the common bile duct (CBD) combined with percutaneous transhepatic cholangiographic drainage (PTCD) and laparoscopic choledocholithotomy with T-tube placement for the treatment of CBD stones. Between January 1991 and July 2002, 50 patients with choledocholithiasis and a CBD diameter larger than or equal to 1 cm underwent laparoscopic CBD explorations. The study group consisted of 10 patients undergoing laparoscopic primary closure of the CBD combined with PTCD. The control group consisted of 40 patients undergoing laparoscopic choledocholithotomy with T-tube placement. Parameters were compared statistically. The study group showed higher female/male ratio (6/4 vs. 8/32, P = 0.02), less stone numbers (1.90 +/- 0.88 vs. 3.40 +/- 1.65, P = 0.0078), shorter operation time (138 +/- 37 minutes vs. 191 +/- 75 minutes, P = 0.014), and shorter postoperative stays (7 +/- 3 days vs. 10 +/- 3 days, P = 0.0013). It seems that laparoscopic primary closure of the CBD combined with PTCD can shorten the operation time and postoperative stays as compared with laparoscopic choledocholithotomy with T-tube placement for the treatment of CBD stones.

Ancillary p16(INK4a) adds no meaningful value to the performance of ER/PR/Vim/CEA panel in distinguishing between primary endocervical and endometrial adenocarcinomas in a tissue microarray study.

PURPOSE: Endocervical adenocarcinomas (ECA) and endometrial adenocarcinomas (EMA) are uterine malignancies that have differing biological behavior. The choice of appropriate therapeutic plan depends indeed on the tumor's site of origin. In this study, we not only compare the individual expression status of five immunomarkers (ER, PR, Vim, CEA, and p16(INK4a)), but also evaluate whether p16(INK4a) adds value to the ER/PR/Vim/CEA panel characteristics in distinguishing between primary ECA and EMA. METHODS: A tissue microarray (TMA) was constructed using paraffin-embedded, formalin-fixed tissues from 35 hysterectomy specimens, including 14 ECA and 21 EMA. TMA sections were immunostained with five anti-bodies, by avidin-biotin complex (ABC) method for antigen visualization. The staining intensity and area extent of the immunohistochemical (IHC) reactions were appraised by using the semi-quantitative scoring system. RESULTS: The four respective markers (ER, PR, Vim, CEA) and their combined panel expressions showed significant (p < 0.05) frequency differences between ECA and EMA tumors. The p16(INK4a) marker also revealed a significant frequency difference (p < 0.05) between the two sites of origin, but did not demonstrate to have any supplementary value to the 4-marker panel. CONCLUSION: According to our data, when there is histomorphological and clinical doubt as to the primary site of origin, we recommend that the conventional 4-marker (ER/PR/Vim/CEA) panel is appropriate. Ancillary p16(INK4a)-marker testing does not add value to the 4-marker panel in distinguishing between primary ECA and EMA.

Multi-center study on patient selection for and the oncologic safety of intraoperative radiotherapy (IORT) with the Xoft Axxent® eBx® System for the management of early stage breast cancer in Taiwan.

BACKGROUND: In this multi-center study, we report the patient selection criteria for and preliminary oncologic outcomes associated with intraoperative radiotherapy (IORT) delivered by the Xoft Axxent® eBx® system for early-stage breast cancer in Taiwan. METHODS: Patients with early breast cancer in Taiwan received breast conserving surgery and received IORT with Xoft Axxent® eBx® System during 2013-2015 was search from database of Taiwan IORT study cooperative group (T-IORTSCG). Patients' clinicopathologic characteristics and early post-operative results were collected and reported. RESULTS: During the study period, 26 hospitals in Taiwan performed a total of 261 Xoft IORT procedures for breast cancer. The mean age of them was 52.9 ± 9.8 years (37-72), and tumor size was 1.5 ± 0.8 cm (0.1-4.2 cm) for invasive cancer and 1.2 ± 0.8 cm (range, 0.2-3.0 cm) for ductal carcinoma in situ (DCIS) lesions. Lymph node metastasis was found in 6 (2.3%) patients. The patients received IORT in Taiwan differed markedly from those used in the ELIOT and TARGIT-A studies. Specifically, patients selected for IORT in Taiwan tended to be younger, their tumors tended to be larger and the prevalence of lymph node metastasis tended to be lower. Among these 261 patients, 8 (3.1%) patients required whole breast radiotherapy. During a mean follow up of 15.6 months, locoregional recurrence was observed in 2 (0.8%) patients. CONCLUSION: In real world experience, patients received IORT differed quite significantly with criteria formulated by trials. The preliminary results of IORT in Taiwan showed it is well acceptable by patients and clinicians.

MicroRNA-30a increases tight junction protein expression to suppress the epithelial-mesenchymal transition and metastasis by targeting Slug in breast cancer.

The epithelial-to-mesenchymal (EMT) transition is a prerequisite for conferring metastatic potential during tumor progression. microRNA-30a (miR-30a) expression was significantly lower in aggressive breast cancer cell lines compared with non-invasive breast cancer and non-malignant mammary epithelial cell lines. In contrast, miR-30a overexpression reversed the mesenchymal appearance of cancer cells to result in a cobblestone-like epithelial phenotype. We identified Slug, one of the master regulators of EMT, as a target of miR-30a using in silico prediction. Reporter assays indicated that miR-30a could bind to the 3'-untranslted region of Slug mRNA. Furthermore, we linked miR-30a to increased expression of claudins, a family of tight junction transmembrane proteins. An interaction between Slug and E-box in the claudin promoter sequences was reduced upon miR-30a overexpression, further leading to reduction of filopodia formation and decreased invasiveness/metastasis capabilities of breast cancer cells. Consistently, delivery of miR-30a in xenografted mice decreased tumor invasion and migration. In patients with breast cancer, a significantly elevated risk of the miR-30alow/CLDN2low/FSCNhigh genotype was observed, linking to a phenotypic manifestation of larger tumor size, lymph node metastasis, and advanced tumor stage among patients. In conclusion, the miR-30a/Slug axis inhibits mesenchymal tumor development by interfering with metastatic cancer cell programming and may be a potential target for therapy in breast cancer.

Foxo3a-mediated overexpression of microRNA-622 suppresses tumor metastasis by repressing hypoxia-inducible factor-1α in ERK-responsive lung cancer.

Metastatic spread of cancer cells portends a poor prognosis and mortality for lung cancer patients. Hypoxia-inducible factor-1α (HIF-1α) enhances tumor cell motility by activating the epithelial-to-mesenchymal transition (EMT), which is considered a prerequisite for metastasis. Recent studies of microRNA involvement in cancer invasion and metastasis have highlighted the role of such RNAs in tumor development. However, little work has been done to identify tumor suppressor microRNAs that target HIF-1α to down-modulate the EMT and thereby counteract the aggressiveness and metastasis of lung cancer cells. Here, we identified the 3'-untranslated region of HIF-1α mRNA as a target of miR-622 and established that miR-622-mediated down-modulation of HIF-1α correlates with decreased levels of mesenchymal proteins, including Snail, ß-catenin, and vimentin. Functional analyses revealed that increased miR-622 expression inhibited lung cancer cell migration and invasion in vitro. miR-622 also inhibited the genesis of metastatic lung nodules as demonstrated in a lung cancer xenograft model in which nude mice were transplanted with A549 cells expressing miR-622. Mechanistic analyses showed that overexpression of EGF decreased the miR-622 level in A549 cells, and this reduction could be rescued by administrating U0126, an inhibitor of ERK. Moreover, miR-622 overexpression mediated by the transcription factor FOXO3a decreased the invasiveness of lung tumor cells by inhibiting HIF-1α via inactivation of ERK signaling in U0126-treated A549 cells. These findings highlight the pivotal role of the FOXO3a/miR-622 axis in inhibiting HIF-1α to interfere with tumor metastasis, and this information may contribute to development of novel therapeutic strategies for treating aggressive lung cancer.

Calcium content of different compositions of gallstones and pathogenesis of calcium carbonate gallstones.

BACKGROUND/OBJECTIVES: Our aim was to investigate the calcium content of different gallstone compositions and the pathogenic mechanisms of calcium carbonate gallstones. METHODS: Between August 2001 and July 2007, gallstones from 481 patients, including 68 calcium carbonate gallstones, were analyzed for total calcium content. Gallbladder bile samples from 33 cases and six controls were analyzed for pH, carbonate anion level, free-ionized calcium concentration and saturation index for calcium carbonate. RESULTS: Total calcium content averaged 75.6 %, 11.8 %, and 4.2 % for calcium carbonate, calcium bilirubinate and cholesterol gallstones. In 29.4 % of patients, chronic and/or intermittent cystic duct obstructions were caused by polypoid lesions in the neck region and 70.6 % were caused by stones. A total of 82 % of patients had chronic low-grade inflammation of the gallbladder wall and 18.0 % had acute inflammatory exacerbations. In the bile, we found the mean pH, mean carbonate anion, free-ionized calcium concentrations, and mean saturation index for calcium carbonate to be elevated in comparison to controls. CONCLUSION: From our study, we found chronic and/or intermittent cystic duct obstructions and low-grade GB wall inflammation lead to GB epithelium hydrogen secretion dysfunction. Increased calcium ion efflux into the GB lumen combined with increased carbonate anion presence increases SI_CaCO(3) from 1 to 22.4. Thus, in an alkaline milieu with pH 7.8, calcium carbonate begins to aggregate and precipitate.

Pathophysiological significance of gallbladder volume changes in gallstone diseases.

AIM: To study the pathophysiological significance of gallbladder volume (GBV) and ejection fraction changes in gallstone patients. METHODS: The fasting GBV of gallstone patients with acute cholecystitis (n = 99), chronic cholecystitis (n = 85) and non-gallstone disease (n = 240) were measured by preoperative computed tomography. Direct saline injection measurements of GBV after cholecystectomy were also performed. The fasting and postprandial GBV of 65 patients with gallstones and chronic cholecystitis and 53 healthy subjects who received health examinations were measured by abdominal ultrasonography. Proper adjustments were made after the correction factors were calculated by comparing the preoperative and postoperative measurements. Pathological correlations between gallbladder changes in patients with acute calculous cholecystitis and the stages defined by the Tokyo International Consensus Meeting in 2007 were made. Unpaired Student's t tests were used. P < 0.05 was deemed statistically significant. RESULTS: The fasting GBV was larger in late stage than in early/second stage acute cholecystitis gallbladders (84.66 +/- 26.32 cm(3), n = 12, vs 53.19 +/- 33.80 cm(3), n = 87, P = 0.002). The fasting volume/ejection fraction of gallbladders in chronic cholecystitis were larger/lower than those of normal subjects (28.77 +/- 15.00 cm(3) vs 6.77 +/- 15.75 cm(3), P < 0.0001)/(34.6% +/- 10.6%, n = 65, vs 53.3% +/- 24.9%, n = 53, P < 0.0001). CONCLUSION: GBV increases as acute cholecystitis progresses to gangrene and/or empyema. Gallstone formation is associated with poorer contractility and larger volume in gallbladders that contain stones.

Distinguishing between primary endocervical and endometrial adenocarcinomas: is a 2-marker (Vim/CEA) panel enough?

Gynecological pathologists are used to operating many panels of various markers in combination for the diagnostic distinction between primary endocervical and endometrial adenocarcinomas. The conventional 3-marker (ER/Vim/CEA) panel is the most promising tool. In this study, our aim is to investigate whether a 2-marker panel is enough to distinguish between these two gynecologic malignancies. Additionally, we wish to determine which one is the most favorable among eight panels tested, including six 2-marker (ER/CEA, PR/CEA, Vim/CEA, ER/p16(INK4a), PR/p16(INK4a), Vim/p16(INK4a)) and two 3-marker (ER/Vim/CEA, ER/Vim/p16(INK)) panels. A tissue microarray was constructed using paraffin-embedded, formalin-fixed tissues from 35 hysterectomy specimens, including 14 primary endocervical adenocarcinomas and 21 primary endometrial adenocarcinomas. Utilizing the avidin-biotin complex (ABC) method, tissue array sections were immunostained with five commercially available antibodies (ER, Vim, CEA, PR, and p16(INK4a)) to evaluate their individual frequencies of expression. We found that all eight aforementioned panels showed an encouraging range of overall accuracy (69.2% to 78.3%). However, one panel of 2-markers (Vim, CEA) exhibited the most efficiency (78.3%) in the diagnostic distinction between primary endocervical and endometrial adenocarcinomas. Based on the analyzed data, we conclude that the 2-marker (Vim/CEA) panel seems adequate to be an appropriate, convenient, and efficient means to distinguish between primary endocervical and endometrial adenocarcinomas. Even though there were a limited number of cases, this study still provides valuable references to help avoid wasting resources and unnecessary marker testing.