Deregulated microRNAs in triple-negative breast cancer revealed by deep sequencing.

BACKGROUND: MicroRNAs (miRNAs) are short, non-coding RNA molecules that play critical roles in human malignancy. However, the regulatory characteristics of miRNAs in triple-negative breast cancer, a phenotype of breast cancer that does not express the genes for estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2, are still poorly understood. METHODS: In this study, miRNA expression profiles of 24 triple-negative breast cancers and 14 adjacent normal tissues were analyzed using deep sequencing technology. Expression levels of miRNA reads were normalized with the quantile-quantile scaling method. Deregulated miRNAs in triple-negative breast cancer were identified from the sequencing data using the Student's t-test. Quantitative reverse transcription PCR validations were carried out to examine miRNA expression levels. Potential target candidates of a miRNA were predicted using published target prediction algorithms. Luciferase reporter assay experiments were performed to verify a putative miRNA-target relationship. Validated molecular targets of the deregulated miRNAs were retrieved from curated databases and their associations with cancer progression were discussed. RESULTS: A novel 25-miRNA expression signature was found to effectively distinguish triple-negative breast cancers from surrounding normal tissues in a hierarchical clustering analysis. We documented the evidence of seven polycistronic miRNA clusters preferentially harboring deregulated miRNAs in triple-negative breast cancer. Two of these miRNA clusters (miR-143-145 at 5q32 and miR-497-195 at 17p13.1) were markedly down-regulated in triple-negative breast cancer, while the other five miRNA clusters (miR-17-92 at 13q31.3, miR-183-182 at 7q32.2, miR-200-429 at 1p36.33, miR-301b-130b at 22q11.21, and miR-532-502 at Xp11.23) were up-regulated in triple-negative breast cancer. Moreover, miR-130b-5p from the miR-301b-130b cluster was shown to directly repress the cyclin G2 (CCNG2) gene, a crucial cell cycle regulator, in triple-negative breast cancer cells. Luciferase reporter assays showed that miR-130b-5p-mediated repression of CCNG2 was dependent on the sequence of the 3'-untranslated region. The findings described in this study implicate a miR-130b-5p-CCNG2 axis that may be involved in the malignant progression of triple-negative breast cancer. CONCLUSIONS: Our work delivers a clear picture of the global miRNA regulatory characteristics in triple-negative breast cancer and extends the current knowledge of microRNA regulatory network.

Cord blood level of insulin-like growth factor-1 and IGF binding protein-3 in monochorionic twins.

BACKGROUND/PURPOSE: Insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) are known to modulate fetal growth but their role in intrauterine growth of monochorionic twins (MCT) has not been studied. METHODS: Cord venous blood was collected directly after birth. IGF-1 and IGFBP-3 in the cord venous blood were quantified by radioimmunoassay. Birth weights (BWs) were obtained electronically. Placentas were examined for chorionicity. RESULTS: Cord blood was collected in 37 pairs of MCT (15 pairs were males). BWs ranged from 564 to 3240 g, and gestational ages (GAs) were between 24 weeks and 39 weeks. There was a correlation between BW and cord venous blood IGFBP-3 concentration (r = 0.28, p = 0.015), but not between BW and cord venous blood IGF-1 level. There was no difference in IGF-1 between the heavier twins (30.8 ± 61.8 ng/mL) and lighter twins (33.2 ± 63.7 ng/mL), but a trend (p = 0.096) of higher IGFBP-3 level was demonstrated in heavier twins (3.14 ± 1.23 µg/mL) than in lighter twins (2.71 ± 1.19 µg/mL). The IGFBP-3 levels were higher (p = 0.042) in female twins (3.20 ± 1.33 µg/mL) than in male twins (2.64 ± 1.04 µg/mL). The IGF-1 level of the heavier twins correlated significantly to their lighter co-twin (r = 0.73, p < 0.001). CONCLUSION: Our data showed that cord venous blood IGF-1 level might be controlled mainly by genetic factors. IGFBP-3 might play an important role in fetal growth.

Multimodel assessment of BRCA1 mutations in Taiwanese (ethnic Chinese) women with early-onset, bilateral or familial breast cancer.

Although evidence suggests an importance of genetic factors in the development of breast cancer in Taiwanese (ethnic Chinese) women, including a high incidence of early-onset and secondary contralateral breast cancer, a major breast cancer predisposition gene, BRCA1, has not been well studied in this population. In fact, the carcinogenic impacts of many genetic variants of BRCA1 are unknown and classified as variants of uncertain significance (VUS). It is therefore important to establish a method to characterize the BRCA1 VUSs and understand their role in Taiwanese breast cancer patients. Accordingly, we developed a multimodel assessment strategy consisting of a prescreening portion and a validated functional assay to study breast cancer patients with early-onset, bilateral or familial breast cancer. We found germ-line BRCA1 mutations in 11.1% of our cohort and identified one novel missense mutation, c.5191C>A. Two genetic variants were initially classified as VUSs (c.1155C>T and c.5191C>A). c.1155C>T is not predicted to be deleterious in the prescreening portion of our assessment strategy. c.5191C>A, on the other hand, causes p.T1691K, which is predicted to have high deleterious probability because of significant structural alteration, a high deleterious score in the predictive programs and, clinically, triple negative characteristics in breast tumors. This mutant is confirmed by transcription activation and yeast growth-inhibition assays. In conclusion, we show as high a prevalence of germ-line BRCA1 mutation in high-risk Taiwanese patients as in Caucasians and demonstrate a useful strategy for studying BRCA1 VUSs.

SPARC (osteonectin) in breast tumors of different histologic types and its role in the outcome of invasive ductal carcinoma.

The purpose of this study was to characterize the immunohistochemical distribution of secreted protein acidic and rich in cystein (SPARC) in benign and malignant breast tumors of different histologic types and define its association with the outcome of invasive ductal carcinoma (IDC) patients. A total of 286 samples of benign and malignant breast lesions between 1994 and 2005 were retrieved from National Taiwan University Hospital. Up to 11 years clinical follow-up data were available for 185 patients with IDC. Immunohistochemistry staining with SPARC was performed in tissue microarray or whole section. The association of expression of SPARC and cumulative overall survival of IDC patients were analyzed using Kaplan-Meier survival analysis and Cox regression analysis. Secreted protein acidic and rich in cystein was not expressed in benign breast phylloides and all benign breast tumors, while expressed in 17.2% of IDC, 85% of metaplastic carcinoma of the breast (MCB), and all malignant breast phylloides. Secreted protein acidic and rich in cystein was strongly expressed in mesenchymal components of MCB and expression levels in epithelial components were variable. The correlation of positive expression of SPARC and poor long-term survival in IDC is significant (p = 0.004). Individuals with positive SPARC expression had 2.34 times higher hazard of death compared with those with negative SPARC expression after adjusting for factors including positive lymph node, TNM tumor stage, estrogen receptor, and progesterone receptor. Secreted protein acidic and rich in cystein may be useful as a prognostic indicator for IDC.

Statistical identification of gene association by CID in application of constructing ER regulatory network.

BACKGROUND: A variety of high-throughput techniques are now available for constructing comprehensive gene regulatory networks in systems biology. In this study, we report a new statistical approach for facilitating in silico inference of regulatory network structure. The new measure of association, coefficient of intrinsic dependence (CID), is model-free and can be applied to both continuous and categorical distributions. When given two variables X and Y, CID answers whether Y is dependent on X by examining the conditional distribution of Y given X. In this paper, we apply CID to analyze the regulatory relationships between transcription factors (TFs) (X) and their downstream genes (Y) based on clinical data. More specifically, we use estrogen receptor alpha (ERalpha) as the variable X, and the analyses are based on 48 clinical breast cancer gene expression arrays (48A). RESULTS: The analytical utility of CID was evaluated in comparison with four commonly used statistical methods, Galton-Pearson's correlation coefficient (GPCC), Student's t-test (STT), coefficient of determination (CoD), and mutual information (MI). When being compared to GPCC, CoD, and MI, CID reveals its preferential ability to discover the regulatory association where distribution of the mRNA expression levels on X and Y does not fit linear models. On the other hand, when CID is used to measure the association of a continuous variable (Y) against a discrete variable (X), it shows similar performance as compared to STT, and appears to outperform CoD and MI. In addition, this study established a two-layer transcriptional regulatory network to exemplify the usage of CID, in combination with GPCC, in deciphering gene networks based on gene expression profiles from patient arrays. CONCLUSION: CID is shown to provide useful information for identifying associations between genes and transcription factors of interest in patient arrays. When coupled with the relationships detected by GPCC, the association predicted by CID are applicable to the construction of transcriptional regulatory networks. This study shows how information from different data sources and learning algorithms can be integrated to investigate whether relevant regulatory mechanisms identified in cell models can also be partially re-identified in clinical samples of breast cancers. AVAILABILITY: the implementation of CID in R codes can be freely downloaded from (http://homepage.ntu.edu.tw/~lyliu/BC/).

Over-expression of EphB3 enhances cell-cell contacts and suppresses tumor growth in HT-29 human colon cancer cells.

Receptor tyrosine kinase EphB3 is expressed in cells in the bottom of intestinal crypts near stem cell niches. Loss of Ephb3 has recently been reported to produce invasive colorectal carcinoma in Apc(Min/+) mice and EphB-mediated compartmentalization was demonstrated to be a mechanism suppressing colorectal cancer progression; however, it is unknown whether other factors contribute to EphB-mediated tumor suppression. EphA4-ephrin-A and EphB4-ephrin-B2 signaling have been reported to promote mesenchymal-to-epithelial transition (MET). Here, we examine whether EphB3-ephrin-B interaction has a similar effect and investigate its role in tumor suppression. We found in a clinical cohort that EphB3 expression was significantly reduced in advanced Dukes' stage tumor specimens, so we over-expressed EphB3 in HT-29 cells by stable transfection. EphB3 over-expression inhibited HT-29 growth in monolayer cultures, anchorage-independent growth in soft agar and xenograft growth in nude mice and initiated morphological, behavioral and molecular changes consistent with MET. Specifically, EphB3 over-expression re-organized cytoskeleton (converting spreading cells to a cobble-like epithelial morphology, patterning cortical actin cytoskeleton and polarizing E-cadherin and ZO-1), induced functional changes favoring MET (decreased transwell migration, increased apoptosis and Ca(2+)-dependent cell-cell adhesion), decreased mesenchymal markers (fibronectin and nuclear beta-catenin), increased epithelial markers (ZO-1, E-cadherin and plakoglobin) and inactivated CrkL-Rac1, a known epithelial-to-mesenchymal transition signaling pathway. Additionally, cross talk from Wnt signaling potentiated the restoration of epithelial cell polarity. Noteworthily, the same factors contributing to MET, owing to EphB3 signaling, also facilitated tumor suppression. We conclude that EphB3-ephrin-B interaction promotes MET by re-establishing epithelial cell-cell junctions and such an MET-promoting effect contributes to EphB3-mediated tumor suppression.

Identification of calreticulin as a prognosis marker and angiogenic regulator in human gastric cancer.

The purpose of this study was to identify genes of interest for a subsequent functional and clinical cohort study using complementary (c)DNA microarrays. cDNA microarray hybridization was performed to identify differentially expressed genes between tumor and nontumor specimens in 30 gastric cancer patients. Subsequent functional studies of the selected gene were carried out, including cell cycle analysis, cell migration analysis, analyses of vascular endothelial growth factor (VEGF) and placenta growth factor (PlGF), and oligo-microarray studies using two pairs of stable cell lines of the selected gene. Another independent cohort study of 79 gastric cancer patients was conducted to evaluate the clinical significance of the selected gene in human gastric cancer. Calreticulin (CRT) was selected for further investigation. Two pairs of stable cell lines of CRT overexpression and CRT knockdown were constructed to perform functional studies. CRT enhanced gastric cancer cell proliferation and migration. Overexpressed CRT upregulated the expression and secretion of PlGF and VEGF. CRT had a reciprocal effect on connective tissue growth factor (CTGF) expression. Positive immunohistochemical staining of calreticulin was significantly correlated with high microvessel density (MVD) (p = 0.014), positive serosal invasion (p = 0.013), lymph node metastasis (p = 0.002), perineural invasion (p = 0.008), and poor patient survival (p = 0.0014). Multivariate survival analysis showed that CRT, MVD, and serosal invasion were independent prognosticators. We conclude that CRT overexpression enhances angiogenesis, and facilitates proliferation and migration of gastric cancer cells, which is in line with the association of CRT with MVD, tumor invasion, lymph node metastasis, and survival in gastric cancer patients.

Preoperative serum placenta growth factor level is a prognostic biomarker in colorectal cancer.

PURPOSE: Increased angiogenesis at the site of the primary tumor in colorectal cancer has been associated with poor prognosis and relapse of disease. We previously demonstrated that the tissue level of placenta growth factor expression was upregulated in colorectal cancer and correlated with disease progression and patient survival. The aims of this study are to examine the prognostic value of serum placenta growth factor, vascular endothelial growth factor, and sFlt-1 and to compare them with the carcinoembryonic antigen levels in patients with colorectal cancer. METHODS: Preoperative serum from 86 patients and serum from 30 healthy controls was included. The levels of sFlt-1, placenta growth factor, vascular endothelial growth factor in the serum were assayed and correlated with the clinical stage results. RESULTS: Serum placenta growth factor, but not vascular endothelial growth factor, increased; sFlt-1 decreased in patients with preoperative colorectal cancer, compared with healthy controls. Higher preoperation serum placenta growth factor levels were associated with higher risk of recurrence. Preoperation serum placenta growth factor, but not carcinoembryonic antigen, was a prognostic indicator in patients with Stage III colorectal cancer. When we use the median level (20.6 pg/ml) of preoperative serum placenta growth factor as a cutoff point, the sensitivity, specificity, and positive predictive value for tumor recurrence and survival was 80, 54, 80% and 70, 56, 70%, respectively. CONCLUSIONS: Preoperative serum placenta growth factor levels were higher in patients with colorectal cancer, were negatively correlated with the serum sFlt-1, and could be used as a prognostic indicator for recurrence and survival for colorectal cancer.

Association between color doppler vascularity index, angiogenesis-related molecules, and clinical outcomes in gastric cancer.

PURPOSE: This study was conducted to evaluate the correlation between color Doppler vascularity index (CDVI), clinical outcomes and five angiogenesis-related molecules including vascular endothelial growth factor (VEGF), placenta growth factor (PlGF), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and calreticulin (CRT) in gastric cancer, and to develop an effective model selected from these five molecules to predict patient survival. PATIENTS AND METHODS: CDVI could be obtained preoperatively by transabdominal ultrasound from 30 patients. Enzyme immunoassay was adopted to determine protein level of VEGF and PlGF, and immunohistochemistry was used to detect COX-2, iNOS and CRT expression. Correlation between CDVI and five individual molecules was assessed. Multiple molecules model was developed using classification and regression tree (CART) analysis from five molecules, and was tested for patient survival in another 45 patients. RESULTS: CDVI was significantly correlated with patient survival (P = 0.00907) and absolute number of metastatic lymph nodes (P = 0.01). There was no significant association between CDVI and any individual molecule. The model, developed by CART consisting of VEGF and PlGF, could differentiate high and low CDVI and survival in testing group (P = 0.00257). CONCLUSIONS: CDVI was associated with lymph node metastasis, combined VEGF and PlGF expression status and patient survival in gastric cancer.

Identification of GRP75 as an independent favorable prognostic marker of neuroblastoma by a proteomics analysis.

PURPOSE: Neuroblastoma (NB) is a heterogeneous neoplasm. Detailed biological discrimination is critical for the effective treatment of this disease. Because the tumor behavior of NB is closely associated with the histologic state of differentiation, we thus aimed to identify novel differentiation-associated markers of NB with prognostic implication. EXPERIMENTAL DESIGN: A human NB cell line SH-SY5Y was used as a model system to explore potential biomarkers for the differentiation of NB by proteomic analyses. Seventy-two NB tumor tissues were subsequently investigated by immunohistochemistry to validate the correlations between the expression of a novel prognostic marker, various clinicopathologic and biological factors, and patient survival. RESULTS: Using two-dimensional differential gel electrophoresis, we found a total of 24 spots of proteins in SH-SY5Y cells whose expression was enhanced following differentiation. Glucose-regulated protein 75 (GRP75) was unambiguously identified as one of the five proteins that were dramatically up-regulated following differentiation. Immunohistochemical analyses of 72 NB tumor tissues further revealed that positive GRP75 immunostaining is strongly correlated with differentiated histologies (P < 0.001), mass-screened tumors (P = 0.016), and early clinical stages (P < 0.001) but inversely correlated with MYCN amplification (P = 0.010). Univariate and multivariate survival analyses showed that GRP75 expression is an independent favorable prognostic factor. CONCLUSIONS: The present findings clearly showed that our proteomics-based novel experimental paradigm could be a powerful tool to uncover novel biomarkers associated with the differentiation of NB. Our data also substantiate an essential role of GRP75 in the differentiation of NB.

Angiopoietin-like protein 1 decreases blood brain barrier damage and edema following focal cerebral ischemia in mice.

Angiopoietin-like protein (Angptl) 1, a member of the angiopoietin-related protein family, modulates angiogenesis but little else is known of its physiological role. We found that angptl1 was upregulated at the 7th day after focal cerebral ischemia in normal mice. In order to understand the role of angptl1 in cerebral infarction, we induced focal cerebral ischemia in normal and glial fibrillary acidic protein promoter-angptl1 transgenic mice. In the transgenic mice without ischemia, overexpression of angptl1 in the whole brain led to a decrease in cortical microvascular density. Following focal cerebral ischemia, edema, but not infarct size, was less in transgenic mice relative to wild type littermates. This effect might be due to a reduction in the blood brain barrier breakdown, as confirmed by a decrease in Evans Blue leakage in the early post-ischemic phase. We conclude that angptl1 may have a beneficial role in the preservation of vascular integrity following focal cerebral ischemia.

Vascularity change and tumor response to neoadjuvant chemotherapy for advanced breast cancer.

For advanced breast cancer with severe local disease (ABC) (stage III/IV), neoadjuvant chemotherapy improves local control and surgical outcome. However, about approximately 20 to 30% of advanced cancers show either no or poor response to chemotherapy. To prevent unnecessary treatment, a capability of predicting clinical response to neoadjuvant chemotherapy of ABC is highly desirable. Vascularity index (VI) of breast cancers was derived from the quantification results in 30 ABC patients by using power Doppler sonography. Power Doppler sonography evaluation was performed every one to two weeks during chemotherapy. The overall response rate for 30 advanced patients tested was 70%, when 50% or more reduction in tumor size was the objective clinical response. Chemotherapy response was unrelated to the original tumor size (p = 0.563) or chemotherapy agents used (p = 0.657). The median VI for all 30 patients was 4.99%. The response rates for hypervascular tumors vs. hypovascular tumors, based on initial median value, were 86.7% and 53.3%, respectively (p = 0.109). The average VIs in responders and nonresponders were 7.67 +/- 4.77% and 4.01 +/- 3.82% (p = 0.052). There was a tendency for responders who have a relatively high initial vascularity. The VI change in responder group shows a pattern of initial increasing in vascularity followed by decreasing in vascularity. All patients (17/17) with a VI increment of >5% during chemotherapy had good chemotherapy response, whereas in patients with a VI increment of <5%, the response rate was 30.8% (4/13) (p < 0.001). For patients with a peak VI of >10% during chemotherapy, the response rate was 94.1% (16/17). However, in patients with a peak VI of <10%, the response rate was 38.5% (5/13) (p = 0.001). This prediction was made mostly within one month (25.47 +/- 12.96 d for VI increments >5% and 25.44 +/- 12.41 d for VI increased to >10%). In the meantime, the differences in size reduction shown in B-mode sonography were insignificant between responders and nonresponders (patient group with VI increment >5%, p = 0.308; patient group with peak VI >10%, p = 0.396). In conclusion, we propose that VI as determined by using power Doppler sonography is a good and inexpensive clinical tool for monitoring vascularity changes during neoadjuvant chemotherapy in ABC patients. Two parameters--VI increment >5% and peak VI >10%--are potential early predictors for good responses to neoadjuvant chemotherapy within one month in patients with ABC.

Facilitating In Vivo Articular Cartilage Repair by Tissue-Engineered Cartilage Grafts Produced From Auricular Chondrocytes.

BACKGROUND: Insufficient cell numbers still present a challenge for articular cartilage repair. Converting heterotopic auricular chondrocytes by extracellular matrix may be the solution. HYPOTHESIS: Specific extracellular matrix may convert the phenotype of auricular chondrocytes toward articular cartilage for repair. STUDY DESIGN: Controlled laboratory study. METHODS: For in vitro study, rabbit auricular chondrocytes were cultured in monolayer for several passages until reaching status of dedifferentiation. Later, they were transferred to chondrogenic type II collagen (Col II)-coated plates for further cell conversion. Articular chondrogenic profiles, such as glycosaminoglycan deposition, articular chondrogenic gene, and protein expression, were evaluated after 14-day cultivation. Furthermore, 3-dimensional constructs were fabricated using Col II hydrogel-associated auricular chondrocytes, and their histological and biomechanical properties were analyzed. For in vivo study, focal osteochondral defects were created in the rabbit knee joints, and auricular Col II constructs were implanted for repair. RESULTS: The auricular chondrocytes converted by a 2-step protocol expressed specific profiles of chondrogenic molecules associated with articular chondrocytes. The histological and biomechanical features of converted auricular chondrocytes became similar to those of articular chondrocytes when cultivated with Col II 3-dimensional scaffolds. In an in vivo animal model of osteochondral defects, the treated group (auricular Col II) showed better cartilage repair than did the control groups (sham, auricular cells, and Col II). Histological analyses revealed that cartilage repair was achieved in the treated groups with abundant type II collagen and glycosaminoglycans syntheses rather than elastin expression. CONCLUSION: The study confirmed the feasibility of applying heterotopic chondrocytes for cartilage repair via extracellular matrix-induced cell conversion. CLINICAL RELEVANCE: This study proposes a feasible methodology to convert heterotopic auricular chondrocytes for articular cartilage repair, which may serve as potential alternative sources for cartilage repair.

Placenta growth factor not vascular endothelial growth factor A or C can predict the early recurrence after radical resection of hepatocellular carcinoma.

The purpose of this study was to evaluate the relationship between the expression of PlGF in tumor tissue and clinical outcomes in HCC patients. Tumor PlGF and vascular endothelial growth factor (VEGF)-A and VEGF-C mRNA were analyzed. Results demonstrated that patients with PlGF expression levels higher than median tended to have early recurrence compared to patients with PlGF expression lower than median (P=.031). In patients with AJCC stage II-III disease, this difference was even more significant (P=.002). In contrast, VEGF-A and VEGF-C could not predict early recurrence-free survival. Since PlGF expression correlated with early recurrence of HCC, PlGF may be an important prognostic indicator in HCC.

Risk prediction for Down's syndrome in young pregnant women using maternal serum biomarkers: determination of cut-off risk from receiver operating characteristic curve analysis.

OBJECTIVE: The aim of this study was to establish a predictive model for Down's syndrome using maternal age as well as maternal serum levels of alpha-fetoprotein (AFP) and human chorionic gonadotropin (hCG), and to identify an optimal cut-off risk in women under the age of 35 years to improve sensitivity. METHODS: Logistic regression models were utilized to predict fetal Down's syndrome as a function of maternal age and logarithm of levels of AFP as well as hCG using training data of 20 pregnancies with fetal Down's syndrome and 9730 unaffected pregnancies. Validation was performed using data of another nine affected pregnancies and 3496 unaffected pregnancies. Receiver operating characteristic (ROC) curves were plotted. RESULTS: Based on the newly established logistic regression equations, the optimal cut-off risk from the ROC curve analysis was at 1:499, with a 17.8% false-positive rate and a 90.0% sensitivity. A suboptimal cut-off risk was estimated at 1:332, with a 12.0% false-positive rate and an 80% sensitivity. CONCLUSION: A predictive model for Down's syndrome was developed using logistic regression. By ROC curve analysis and clinical consideration, the cut-off risk for young pregnant women could be determined.

Outcome for fetuses with prenatally detected congenital heart disease and cardiac arrhythmias in Taiwan.

BACKGROUND/PURPOSE: Outcome for fetuses with prenatally detected congenital heart disease (CHD) and/or cardiac arrhythmias is important for prenatal counseling and perinatal management; however, there exists little literature regarding the outcome for CHD diagnosed in utero in Taiwan. Therefore, we attempted to investigate the outcome for fetuses with CHD and/or cardiac arrhythmias diagnosed prenatally at a tertiary care medical center in Taiwan. METHODS: Between January 1995 and December 2000, 339 patients referred to the National Taiwan University Hospital for fetal echocardiography were included in this study. Medical records were reviewed retrospectively to determine the salient clinical characteristics for all fetuses. RESULTS: CHD was found in 103 fetuses. Gestational age at diagnosis ranged from 17 to 40 weeks; in 37 cases (35.9%) the diagnosis was made before 24 weeks. Mean gestational age at diagnosis was 27.8 weeks. Of the 103 cases, 15 fetuses (14.6%) had major extra cardiac malformations and 15 fetuses (14.6%) had chromosomal abnormalities (five had both) and 30 pregnancies (29.1%) were terminated. Of the remaining 73 pregnancies, three (4.1%) of the fetuses died in utero and 28 (38.4%) postnatally, with 42 (57.5%) surviving. The mortality rates were both 60% in cases with extracardiac or chromosomal anomalies. Arrhythmias were identified in 25, and two pregnancies involving hydrops fetalis were terminated. Of the remaining 23 continued pregnancies, two (8.7%) with long QT syndrome expired postnatally. CONCLUSION: Outcome for fetuses with prenatally detected CHD remains poor, with the prognosis negatively influenced by the presence of complex heart defects as well as extracardiac and chromosomal anomalies. However, prognosis is good for fetuses with cardiac arrhythmia, except with long QT syndrome or hydrops fetalis.

Single-Stage Cartilage Repair Using Platelet-Rich Fibrin Scaffolds With Autologous Cartilaginous Grafts.

BACKGROUND: To avoid complicated procedures requiring in vitro chondrocyte expansion for cartilage repair, the development of a culture-free, 1-stage approach combining platelet-rich fibrin (PRF) and autologous cartilage grafts may be the solution. PURPOSE: To develop a feasible 1-step procedure to combine PRF and autologous cartilage grafts for articular chondral defects. STUDY DESIGN: Controlled laboratory study Methods: The chemotactic effects of PRF on chondrocytes harvested from the primary culture of rabbit cartilage were evaluated in vitro and ex vivo. The rabbit chondrocytes were cultured with different concentrations of PRF media and evaluated for their cell proliferation, chondrogenic gene expression, cell viability, and extracellular matrix synthesis abilities. For the in vivo study, the chondral defects were created on established animal models of rabbits. The gross anatomy, histology, and objective scores were evaluated to validate the treatment results. RESULTS: PRF improved the chemotaxis, proliferation, and viability of the cultured chondrocytes. The gene expression of the chondrogenic markers, including type II collagen and aggrecan, revealed that PRF induced the chondrogenic differentiation of cultured chondrocytes. PRF increased the formation and deposition of the cartilaginous matrix produced by cultured chondrocytes. The efficacy of PRF on cell viability was comparable with that of fetal bovine serum. In animal disease models, morphologic, histological, and objectively quantitative evaluation demonstrated that PRF combined with cartilage granules was feasible in facilitating chondral repair. CONCLUSION: PRF enhances the migration, proliferation, viability, and differentiation of chondrocytes, thus showing an appealing capacity for cartilage repair. The data altogether provide evidence to confirm the feasibility of 1-stage, culture-free method of combining PRF and autologous cartilage graft for repairing articular chondral defects. CLINICAL RELEVANCE: The single-stage, culture-free method of combining PRF and autologous cartilage is useful for repairing articular chondral defects. These advantages benefit clinical translation by simplifying and potentiating the efficacy of autologous cartilage transplantation.

Identification of forty-five novel and twenty-three known NF1 mutations in Chinese patients with neurofibromatosis type 1.

Neurofibromatosis type 1 (NF1), characterized by skin neurofibromas and an excess of café-au-lait spots, is due to mutations in the neurofibromin (NF1) gene. Identifying the genetic defect in individuals with the disease represents a significant challenge because the gene is extremely large with a high incidence of sporadic mutations across the entire gene ranging from single nucleotide substitutes to large deletions. In the present study, we have used a combination of techniques (heteroduplex analysis, sequencing, loss of heterozygosity and quantification of gene dosage) to define the genetic defect in 68 individuals from a cohort of 107 NF1 Taiwanese patients of Chinese origin. Fifty-eight were initially identified using heteroduplex analytical techniques and confirmed by sequence analysis. A further five were identified by direct sequence analysis alone. The reminders were shown to carry large deletions in the NF1 gene by demonstrating loss of heterozygosity that was confirmed by gene dosage measurements using quantitative-PCR techniques. Mis-sense, non-sense, frame-shift or splice-site mutations were identified across the entire gene of which the majority (45/68) were novel in nature. The detection rate with the various analytical techniques and the types of mutation detected are consistent with published data involving both individuals and large cohort studies from other ethnic backgrounds.

Gene expression profile predicts patient survival of gastric cancer after surgical resection.

PURPOSE: This study was conducted to characterize gene expression profile of survival in patients with surgically curable gastric cancer by using an in-house membrane microarray and developing a survival prediction model. MATERIALS AND METHODS: Data of cDNA microarrays were obtained from 18 pairs of cancerous and noncancerous gastric tissues. Nine patients who survived > 30 months were identified as good survival, and the other nine, who survived < 12 months, were identified as poor survival. Supervised analysis was performed to identify a gene expression profile by good and poor survival. Semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) was used to confirm the microarray data in 10 patients with sufficient RNA. Using these 10 patients and another 10 patients selected randomly from 40 newly enrolled patients as the training group, the RT-PCR status of the confirmed genes was used for predicting good versus poor survival. Finally, the prediction model was tested in the remaining 30 newly enrolled gastric cancer patients. RESULTS: A survival prediction model consisting of three genes (CD36, SLAM, PIM-1) was developed. This model could correctly predict poor or good survival in 23 (76.7%) of 30 newly enrolled patients, and yielded a specificity of 80% and a sensitivity of 73.3%. The survival rate of the patients predicted to have good survival was significantly higher than that of those predicted to have poor survival in the test group as a whole (N = 30; P = .00531) and in stage III patients (n = 16; P = .04467). CONCLUSION: The semiquantitative RT-PCR gene expression profiling of three genes extracted from microarray study can accurately predict surgery-related outcome in gastric cancer patients.

Erratum to: Denaturing high-performance liquid chromatography screening of the long QT syndrome-related cardiac sodium and potassium channel genes and identification of novel mutations and single nucleotide polymorphisms.

The name Fon-Jou Hsieh was inadvertently omitted from the list of authors. The name should be added as the third author of the article.