[Acute ischemic stroke and troponin elevation: update of the Mannheim clinical algorithm]. / Akuter ischämischer Schlaganfall und erhöhter Troponinwert ­ Update des Mannheimer Algorithmus.

Elevated high-sensitivity cardiac troponin (hs-cTn) levels should be expected in about half of all patients with acute ischemic stroke (AIS). Since those patients are at risk of increased morbidity and mortality, often attributable to cardiac causes, an adequate work-up of the underlying etiology is required. This can only be achieved by a team of cardiologists and neurologists. Since underlying causes of hs-cTn elevation in AIS patients are diverse, often atypical or silent in their clinical presentation and some, such as an accompanying myocardial infarction, can be acutely life-threatening, the work-up should follow a standardized clinical algorithm. The vast majority of hs-cTn elevations are caused by non-ischemic myocardial injury associated with AIS. This work presents a practice-oriented approach to differential diagnosis with the update of the Mannheim clinical algorithm for acute ischemic stroke and troponin elevation.

Streamlining Acute Abdominal Aortic Dissection Management-An AI-based CT Imaging Workflow.

Life-threatening acute aortic dissection (AD) demands timely diagnosis for effective intervention. To streamline intrahospital workflows, automated detection of AD in abdominal computed tomography (CT) scans seems useful to assist humans. We aimed at creating a robust convolutional neural network (CNN)-based pipeline capable of real-time screening for signs of abdominal AD in CT. In this retrospective study, abdominal CT data from AD patients presenting with AD and from non-AD patients were collected (n 195, AD cases 94, mean age 65.9 years, female ratio 35.8%). A CNN-based algorithm was developed with the goal of enabling a robust, automated, and highly sensitive detection of abdominal AD. Two sets from internal (n = 32, AD cases 16) and external sources (n = 1189, AD cases 100) were procured for validation. The abdominal region was extracted, followed by the automatic isolation of the aorta region of interest (ROI) and highlighting of the membrane via edge extraction, followed by classification of the aortic ROI as dissected/healthy. A fivefold cross-validation was employed on the internal set, and an ensemble of the 5 trained models was used to predict the internal and external validation set. Evaluation metrics included receiver operating characteristic curve (AUC) and balanced accuracy. The AUC, balanced accuracy, and sensitivity scores of the internal dataset were 0.932 (CI 0.891-0.963), 0.860, and 0.885, respectively. For the internal validation dataset, the AUC, balanced accuracy, and sensitivity scores were 0.887 (CI 0.732-0.988), 0.781, and 0.875, respectively. Furthermore, for the external validation dataset, AUC, balanced accuracy, and sensitivity scores were 0.993 (CI 0.918-0.994), 0.933, and 1.000, respectively. The proposed automated pipeline could assist humans in expediting acute aortic dissection management when integrated into clinical workflows.

Artificial Intelligence-Based Emphysema Quantification in Routine Chest Computed Tomography: Correlation With Spirometry and Visual Emphysema Grading.

OBJECTIVE: The aim of the study is to assess the correlation between artificial intelligence (AI)-based low attenuation volume percentage (LAV%) with forced expiratory volume in the first second to forced vital capacity (FEV1/FVC) and visual emphysema grades in routine chest computed tomography (CT). Furthermore, optimal LAV% cutoff values for predicting a FEV1/FVC < 70% or moderate to more extensive visual emphysema grades were calculated. METHODS: In a retrospective study of 298 consecutive patients who underwent routine chest CT and spirometry examinations, LAV% was quantified using an AI-based software with a threshold < -950 HU. The FEV1/FVC was derived from spirometry, with FEV1/FVC < 70% indicating airway obstruction. The mean time interval of CT from spirometry was 3.87 ± 4.78 days. Severity of emphysema was visually graded by an experienced chest radiologist using an established 5-grade ordinal scale (Fleischner Society classification system). Spearman correlation coefficient between LAV% and FEV1/FVC was calculated. Receiver operating characteristic determined the optimal LAV% cutoff values for predicting a FEV1/FVC < 70% or a visual emphysema grade of moderate or higher (Fleischner grade 3-5). RESULTS: Significant correlation between LAV% and FEV1/FVC was found (ϱ = -0.477, P < 0.001). Increasing LAV% corresponded to higher visual emphysema grades. For patients with absent visual emphysema, mean LAV% was 2.98 ± 3.30, for patients with trace emphysema 3.22 ± 2.75, for patients with mild emphysema 3.90 ± 3.33, for patients with moderate emphysema 6.41 ± 3.46, for patients with confluent emphysema 9.02 ± 5.45, and for patients with destructive emphysema 16.90 ± 8.19. Optimal LAV% cutoff value for predicting a FEV1/FVC < 70 was 6.1 (area under the curve = 0.764, sensitivity = 0.773, specificity = 0.665), while for predicting a visual emphysema grade of moderate or higher, it was 4.7 (area under the curve = 0.802, sensitivity = 0.766, specificity = 0.742). Furthermore, correlation between visual emphysema grading and FEV1/FVC was found. In patients with FEV1/FVC < 70% a high proportion of subjects had emphysema grade 3 (moderate) or higher, whereas in patients with FEV1/FVC ≥ 70%, a larger proportion had emphysema grade 3 (moderate) or lower. The sensitivity for visual emphysema grading predicting a FEV1/FVC < 70% was 56.3% with an optimal cutoff point at a visual grade of 4 (confluent), demonstrating a lower sensitivity compared with LAV% (77.3%). CONCLUSIONS: A significant correlation between AI-based LAV% and FEV1/FVC as well as visual CT emphysema grades can be found in routine chest CT suggesting that AI-based LAV% measurement might be integrated as an add-on functional parameter in the evaluation of chest CT in the future.

Influence of contrast agent on artificial intelligence-based CT low attenuation volume percentage measurement.

BACKGROUND: Low attenuation volume percentage (LAV%) has been identified as a quantitative imaging biomarker for emphysema with good correlation with spirometry. The influence of intravenous contrast agent on LAV% and its correlation with spirometry is not well known. PURPOSE: To evaluate the influence of intravenous contrast agent on artificial intelligence (AI)-based LAV% in correlation with spirometric Tiffeneau-Pinelli Index (TI). MATERIAL AND METHODS: In a retrospective study, two groups of 47 patients (mean age 68.04 ± 12.64 and 67.89 ± 11.54 years) with either non-enhanced chest computed tomography (CT) or contrast-enhanced CT were compared. Using an AI-based software, LAV% was quantified using a threshold <-950 HU. TI was calculated from spirometry and pathologic airway obstruction was considered with a TI <70. The effect of contrast agent on LAV% and the relationship between TI and LAV% was analyzed. Correlation coefficients between TI and LAV% were compared for both groups. RESULTS: Patients with non-enhanced CT had a mean LAV% of 9.07 ± 7.53. Of them, 22 patients had a TI <70% and 25 patients a TI ≥70%. Patients with contrast-enhanced CT had a mean LAV% of 6.54 ± 4.62. Of them, 20 patients had a TI <70% and 27 patients had a TI ≥70%. Contrast agent did not show a major effect on LAV% (P = 0.099) and the relationship between TI and LAV% (P = 0.88). In both groups, a significant correlation between TI and LAV% was found (ρ = -0.317 for non-enhanced CT; ρ = -0.514 for contrast-enhanced CT). Difference between correlation coefficients was insignificant. CONCLUSION: Our findings suggest that contrast agent does not influence LAV% nor its correlation with TI.

Quantitative analysis of coronary plaque composition by dual-source CT in patients with acute non-ST-elevation myocardial infarction compared to patients with stable coronary artery disease correlated with virtual histology intravascular ultrasound.

RATIONALE AND OBJECTIVES: To quantitatively assess coronary atherosclerotic plaque composition in patients with acute non-ST elevation myocardial infarction (NSTEMI) and patients with stable coronary artery disease (CAD) by coronary computed tomography angiography (cCTA) correlated with virtual histology intravascular ultrasound (VH-IVUS). MATERIALS AND METHODS: Sixty patients (35 with NSTEMI) were included. Corresponding plaques were assessed by dual-source cCTA and VH-IVUS regarding volumes and percentages of fatty, fibrous, and calcified component; overall plaque burden; and maximal percent area stenosis. Possible differences between patient groups were investigated. Concordance between cCTA and VH-IVUS measurements was validated by Bland-Altman analysis. RESULTS: Forty corresponding plaques (22 of patients with NSTEMI) were finally analyzed by cCTA and VH-IVUS. cCTA plaque analysis revealed no significant differences between plaques of patients with NSTEMI and stable CAD regarding absolute and relative amounts of any plaque component (fatty: 20 mm³/13% versus 17 mm³/14%; fibrous: 81 mm³/63% versus 80 mm³/53%; calcified: 16 mm³/14% versus 26 mm³/26%; all P > .05) or overall plaque burden (153 mm³ versus 165 mm³; P > .05), nor did VH-IVUS plaque analysis. VH-IVUS measured a higher area stenosis in patients with NSTEMI compared to patients with stable CAD (76% versus 68%, P = .01; in cCTA 69% versus 65%, P = .2). Volumes of fatty component were measured systematically lower in cCTA, whereas calcified and fibrous volumes were higher. No significant bias was observed comparing volumes of overall noncalcified component and overall plaque burden. CONCLUSION: Plaques of patients with acute NSTEMI and of patients with stable CAD cannot be differentiated by quantification of plaque components. cCTA and VH-IVUS differ in plaque component analysis.

Evaluation of the Metabolic Response to Cyclopamine Therapy in Pancreatic Cancer Xenografts Using a Clinical PET-CT System.

OBJECTIVES: We analyzed the effects of anti-hedgehog signaling on the (18)F-FDG uptake of pancreatic cancer xenografts (PCXs) using a clinically implemented positron emission tomography (PET)-computer tomography (CT) scanner with high-resolution reconstruction. METHODS: PCXs from two pancreatic cancer cell lines were developed subcutaneously in nude mice and injected intraperitoneally with a low dose of cyclopamine for 1 week. (18)F-FDG PET-CT was performed using a new-generation clinical PET-CT scanner with minor modifications of the scanning protocol to adapt for small-animal imaging. The data set was reconstructed and quantified using a three-dimensional workstation. RESULTS: MiaPaCa-2 cells, which respond to cyclopamine, showed decreased (18)F-FDG uptake without a change in tumor size. For hip tumors, the maximum standardized uptake value (SUV(max)) was reduced by -24.5 ± 9.2%, the average SUV (SUV(avg)) by -33.5 ± 7.0%, and the minimum SUV (SUV(min)) by -54.4 ± 11.5% (P < .05). For shoulder tumors, SUV(max) was reduced by -14.7 ± 7.5%, SUV(avg) by -12.6 ± 6.3, and SUV(min) by -30.3 ± 16.7% (P < .05). Capan-1 cells, which do not respond to cyclopamine, did not show significant SUV changes. CONCLUSIONS: The new generations of clinically implemented PET-CT scanners with high-resolution reconstruction detect a minimal response of PCX to low-dose short-term cyclopamine therapy without changes in tumor size and offer potential for preclinical translational imaging.

Biomarkers vs conventional histological analysis to detect lymph node micrometastases in bladder cancer: a real improvement?

UNLABELLED: Study Type - Therapy (case series) Level of Evidence 4. What's known on the subject? and What does the study add? Muscle invasive bladder cancer has a mortality rate at 5 years of 50%, despite radical therapy, as a result of tumour progression and dissemination. This suggests that half of patients have disseminated disease at the time of diagnosis, which is not detected by the staging techniques currently used. The prognostic factors (histological grade and tumour stage) and current staging techniques do not discriminate between those patients who will be cured with surgical treatment and those who will die from metastatic spread. New diagnostic and prognostic tools that complement the existing methods and provide a proper assessment of carcinoma invading bladder muscle are therefore essential. Molecular staging techniques using specific biomarkers have been applied in various solid tumours to determine the presence of missed tumour cells in lymph nodes (LNs) during routine pathological examination. These techniques could identify patients with LN micrometastases who may potentially benefit from early treatment with chemotherapy. This study compares the performance of conventional histological analysis and molecular biomarkers in detecting bladder cancer LN micrometastases and predicting patient's clinical outcome. The study found that, even though a clear trend to a worse outcome was shown in those patients who became node-positive after molecular analysis, no statistical differences were found in cancer-specific and recurrence-free survival analysis between those patients who were negative by histology but positive by molecular analysis and those who were negative by both techniques. We concluded that molecular analysis of LN spreading in bladder cancer has a better detection rate than conventional histological examination. OBJECTIVE: • To improve the sensitivity of histological examination in detecting occult lymph node (LN) dissemination of bladder cancer using gene expression analysis. PATIENTS AND METHODS: • We carried out a retrospective study that included 504 formalin-fixed paraffin-embedded LNs from 90 patients with muscle invasive bladder cancer and 35 controls. • Gene expression values of two molecular biomarkers (FXYD3 and KRT20) were analysed using reverse transcription real-time quantitative PCR (RT-qPCR). • Molecular results were compared with histological status and patients' clinical outcomes. RESULTS: • Of the 90 patients analysed, 16 were positive and 74 were negative by histological analysis. Of these 74, 19 were classified as positive using RT-qPCR. • Significant differences in cancer-specific (P= 0.011) and recurrence-free (P= 0.009) survival were found between the three patient groups (patients positive by both techniques, patients negative by both techniques, and patients negative by histological but positive by molecular analysis). • A significant difference was not found between histologically negative but molecularly positive patients and patients who were negative by both techniques, but a clear trend to a worse outcome was found in those patients who became node-positive after molecular analysis. CONCLUSIONS: • The analysis of FXYD3 and KRT20 could improve current pathological examination for the detection of micrometastases in LNs. • Further and more extensive studies will determine the real prognostic value of such LN micrometastases.

Two non-invasive GFR-estimation methods in rat models of polycystic kidney disease: 3.0 Tesla dynamic contrast-enhanced MRI and optical imaging.

BACKGROUND: The aim of this study was the assessment of kidney morphology and glomerular filtration rate (GFR) in rat models of polycystic kidney disease and a healthy control group of Sprague-Dawley rats (SD rats). The performance of two non-invasive GFR estimation methods-3.0 Tesla magnetic resonance imaging (MRI) and optical imaging were investigated. Data of GFR assessment was compared to surrogate markers of kidney function and renal histology. METHODS: Optical imaging of GFR was performed transcutaneously in a small animal imaging system with the fluorescent renal marker fluorescein-isothiocyanate-labelled-sinistrin. Morphologic and dynamic renal imaging was done on a clinical 3.0T MR scanner. Renal perfusion analysis was performed with a two-compartment filtration model. RESULTS: The healthy SD rats showed physiological levels of creatinine and urea, indicating normal kidney function. These parameters were elevated in the small animal groups of polycystic kidney disease. For the calculation of perfusion and filtration parameters of kidney function in MRI, a 2D turbo FLASH sequence was performed and allowed to distinguish between normal GFR of healthy rats and reduced GFR of rats with polycystic kidney disease. Also, MRI GFR varied among two different rat strains of polycystic kidney disease, according to their status of renal function impairment. Optical imaging GFR confirmed higher GFR values in healthy rats compared to ill rats but did not show different results among the two rat strains of polycystic kidney disease. For this reason, MRI and optical imaging GFR estimation presented an intra-method bias. CONCLUSIONS: Both non-invasive estimation methods of GFR, MRI and optical imaging, can differentiate between healthy rats and animals with limited kidney function. Furthermore, optical imaging, unlike MRI, seems to consider that disease progression with increase of renal polycystic deterioration does not correlate with decrease of GFR in the initial stage of compensatory hyperfiltration.

Attenuation-based characterization of coronary atherosclerotic plaque: comparison of dual source and dual energy CT with single-source CT and histopathology.

OBJECTIVE: To compare different CT acquisition techniques regarding for attenuation-based characterization of coronary atherosclerotic plaques using histopathology as the standard of reference. MATERIALS AND METHODS: In a post mortem study 17 human hearts were studied with dual-source CT (DSCT) and dual energy CT (DECT) mode on a DSCT as well as with 16-slice single-source CT (SSCT). At autopsy, atherosclerotic lesions were cut at 5 µm sections. Histopathologic classification of the plaques according to the American Heart Association (AHA) criteria was performed by two pathologists. Attenuation values of all plaques were measured in DSCT, DECT and SSCT studies, respectively and classified based on attenuation according to modified AHA criteria. RESULTS: 58 coronary plaques were identified at autopsy. Regardless of the CT technique only 52/58 plaques were found at CT (sensitivity=89.6%). There was no significant difference between the mean attenuation values of different plaque types between DSCT, DECT, and SSCT: type IV: 11HU/8HU/19HU; type Va: 44HU/45HU/52HU; type Vb: 1088HU/966HU/1079HU). The sensitivity for correct classification varied depending on the plaque type (type II=0%, type III=0%, type IV=43%, type Va=58%, Vb=97%). CONCLUSION: Independent of the used acquisition technique, SSCT, DSCT and DECT show similar results for attenuation-based characterization of atherosclerotic coronary plaques.

Expression and significance of FXYD-3 protein in gastric adenocarcinoma.

OBJECTIVE: FXYD-3, also known as Mat-8, is a member of the FXYD protein family. It was reported that this protein can associate with and modify the transport properties of Na, K-ATPase, and may play an important role in a variety of physiological and pathological states. This protein is up-regulated in certain types of cancers (such as breast, prostate and pancreatic cancer), but down-regulated in other types of cancers (such as colon and kidney cancer). No study has been performed in gastric cancer; therefore, the aim of this project was to investigate FXYD-3 expression and its clinicopathological significance in gastric adenocarcinoma. PATIENTS AND METHODS: FXYD-3 protein was examined by immunohistochemistry in normal gastric mucous (n= 29) and gastric adenocarcinoma (n=51), obtained from surgical resection of gastric cancer patients. RESULTS: FXYD-3 protein was present in the cytoplasm of normal gastric epithelial cells or gastric cancer cells. The rate of FXYD-3 strong expression was significantly higher in cancer (51% of 51) than in normal mucosa (10% of 29, X;{2}=13.210, p < 0.0001). FXYD-3 expressed strongly in ulcerative/infiltrating types of cancers compared to polypoid/fungating ones (X;{2}=5.765, p=0.016). However, FXYD-3 expression was not correlated with patient's gender, age, tumor size, lymph node status and histological grade (p > 0.05). Conclosion: Up-regulated expression of FXYD-3 protein may be involved in tumourgenesis and invasion of gastric adenocarcinoma.

Expression of FXYD3 protein in relation to biological and clinicopathological variables in colorectal cancers.

BACKGROUND: FXYD3 is up-/down-regulated in different types of cancers. We examined FXYD3 expression in colorectal cancers and its relationship to biological and clinicopathological variables. PATIENTS AND METHODS: Expression of FXYD3 protein was immunohistochemically examined in distant normal mucosa (n = 34), adjacent normal mucosa (n = 72), primary tumour (n = 150) and lymph node metastasis (n = 35) from colorectal cancer patients. RESULTS: FXYD3 was highly expressed in primary tumour compared to adjacent normal mucosa (p = 0.02). FXYD3 was or tended to be positively related to the expression of ras (p = 0.02), p53 (p = 0.06), legumain (p = 0.02) and proliferating cell nuclear antigen (p = 0.03). Moreover, there was a higher frequency of strong FXYD3 expression in Dukes A-C tumours than in D tumours (p = 0.04). The strong FXYD3 expression tended to predict worse survival in the patients with Dukes A + B tumour (p = 0.07), while there was no such tendency in the patients with Dukes C + D tumour (p = 0.94). The tumours located in the colon had a higher degree of FXYD3 expression than the tumours located in the rectum (p = 0.05). CONCLUSION: The FXYD3 was associated with certain biological variables and may be involved in the development of the relative earlier stages of colorectal cancers.

Expression of FXYD-3 is an independent prognostic factor in rectal cancer patients with preoperative radiotherapy.

PURPOSE: FXYD-3 (MAT-8) is overexpressed in several types of cancers; however, its clinical relevance in rectal cancers has not been studied. Therefore, we examined FXYD-3 expression in rectal cancers from the patients who participated in a Swedish clinical trial of preoperative radiotherapy (RT) to determine whether FXYD-3 was overexpressed in rectal cancers and correlated with RT, survival, and other clinicopathologic variables. METHODS AND MATERIALS: The study included 140 rectal cancer patients who participated in a clinical trial of preoperative RT, 65 with and 75 without RT before surgery. FXYD-3 expression was immunohistochemically examined in distant (n = 70) and adjacent (n = 101) normal mucosa, primary tumors (n = 140), and lymph node metastasis (n = 36). RESULTS: In the whole cohort, strong FXYD-3 expression was correlated with infiltrative tumor growth (p = 0.02). In the RT group, strong FXYD-3 expression alone (p = 0 .02) or combined with phosphatase of regenerating liver was associated with an unfavorable prognosis (p = 0.02), independent of both TNM stage and tumor differentiation. In tumors with strong FXYD-3 expression, there was less tumor necrosis (p = 0.02) and a trend toward increased incidence of distant metastasis (p = 0.08) after RT. None of these effects was seen in the non-RT group. FXYD-3 expression in the primary tumors tended to be increased compared with normal mucosa regardless of RT. CONCLUSION: FXYD-3 expression was a prognostic factor independent of tumor stage and differentiation in patients receiving preoperative RT for rectal cancer.

Identification of malignancy factors by analyzing cystic tumors of the pancreas.

AIM: The diversity in the aggressiveness of cystic tumors of the pancreas - ranging from the usually benign serous cystadenoma to lesions of variable degrees of malignancy - was utilized for the identification of molecular factors that are involved in the occurrence of malignancy. METHODS: We analyzed the transcript profiles of different cystic tumor types. The results were confirmed at the protein level by immunohistochemistry. Also, functional studies with siRNA silencing were performed. RESULTS: Expression variations at the RNA and protein level were identified that are closely correlated with the degree of malignancy. Besides, all tumors could be classified effectively by this means. Many of the identified factors had not previously been known to be associated with malignant cystic lesions. siRNA silencing of the gene with the most prominent variation - the anti-apoptotic factor FASTK (Fas-activated serine/threonine kinase) - revealed a regulative effect on several genes known to be relevant to the development of tumors. CONCLUSION: By a molecular analysis of rare types of pancreatic cancer, which are less frequent in terms of disease, variations could be identified that could be critical for the regulation of malignancy and thus relevant to the treatment of also the majority of pancreatic tumors.

FXYD3 expression in gliomas and its clinicopathological significance.

FXYD3, interacting with Na+/K+-ATPase, is considered a cell surface regulator modulating the function of ion pumps and ion channels. The FXYD3 gene was originally cloned from murine mammary tumors and then from human breast tumors. However, no study of FXYD3 has been carried out in gliomas; therefore, we examined FXYD3 expression in gliomas and its clinicopathological significance. FXYD3 expression was immunohistochemically examined in 71 primary gliomas, along with 37 matched adjacent normal brain samples and 8 recurred gliomas. The frequency of strong FXYD3 expression was higher in the primary tumors in either unmatched (p = 0.046) or matched cases (p = 0.02), compared to normal brain tissue. FXYD3 expression was significantly more increased in females than males (p = 0.01), and in multiple site gliomas than single sites (p = 0.02). There was no difference of FXYD3 expression regarding age, tumor location, size, histological type, and tumor grade (p > 0.05). The results suggest that FXYD3 expression may be involved in glioma development, especially in multiple gliomas and female patients.

Expression of the Shwachman-Bodian-Diamond syndrome (SBDS) protein in human pancreatic cancer and chronic pancreatitis.

BACKGROUND: The Shwachman-Bodian-Diamond syndrome (SBDS) protein is a member of a highly conserved family which influences RNA activation and is associated with pancreatic, skeletal and bone marrow deficiencies, as well as hematological malignancies. METHODS: In this study, the expression and localization of SBDS were investigated in normal human pancreatic tissues, chronic pancreatitis (CP) tissues, primary and metastatic pancreatic ductal adenocarcinoma (PDAC) tissues, as well as in cultured pancreatic cancer cell lines by immunohistochemistry, immunoblotting and immunocytochemistry. RESULTS: In the normal pancreas, SBDS was localized in the cytoplasm of islet cells and ductal cells. In CP tissues, SBDS was found in the cytoplasm of ductal cells, tubular complexes, stromal fibroblasts and in PanIN1-2 lesions. In PDAC tissues, SBDS exhibited cytoplasmic and occasionally nuclear localization in tubular complexes, PanIN1-3 lesions, cancer cells, and stromal fibroblasts. Different levels of SBDS protein were detected in cultured pancreatic cancer cell lines. CONCLUSION: SBDS is expressed in normal, CP, and PDAC tissues, as well as in pancreatic cancer cell lines. The different expression and localization patterns suggest a role of SBDS in the pathogenesis of, or response to, inflammatory and neoplastic pancreatic diseases.

Loss of ONECUT1 expression in human pancreatic cancer cells.

ONECUT1 (HNF-6) is the prototype of a new class of homeodomain transcription factors, that controls the development of pancreatic ducts during mouse development. In the present study, the role of ONECUT1 and its targeted genes TCF2, PKHD1 and CYS1 was analyzed in human pancreatic ductal adenocarcinoma (PDAC). mRNA levels of ONECUT1, TCF2, PKHD1 and CYS1 were measured in pancreatic tissues and pancreatic cancer cell lines by quantitative reverse-transcriptase polymerase chain reaction (QRT-PCR). Protein expression of ONECUT1 and TCF2 was assessed in pancreatic tissues by immunohistochemistry. ONECUT1 was transfected into Panc-1 and T3M4 pancreatic cancer cells and its effects on anchorage-dependent and -independent growth as well as invasion and adhesion were analyzed. Median mRNA levels of ONECUT1, TCF2, PKHD1 and CYS1 were 7.7-, 2.0-, 5.7- and 3.8-fold higher in normal tissues than in PDAC tissues. ONECUT1 protein was expressed in normal acinar and ductal cells, but neither in the cancer cells of PDAC tissues nor in 7 of 8 cultured pancreatic cancer cell lines. There was a significant positive correlation between ONECUT1 and TCF2, CYS1, and PKHD1 mRNA levels in PDAC tissues. Transfection of ONECUT1 into pancreatic cancer cells resulted in up-regulation of the target gene TCF2, a reduction in invasiveness, but no change in adhesion or growth. In conclusion, ONECUT1 expression is lost in pancreatic cancer cells, suggesting a tumor suppressor function in this malignancy.

Adrenomedullin is induced by hypoxia and enhances pancreatic cancer cell invasion.

Adrenomedullin (ADM) is synthesized by different types of cells and acts by binding calcitonin receptor-like receptor (CRLR) and members of the receptor activity-modifying protein (RAMP) family. In this study, the expression and functional role of ADM and its signaling components were investigated in pancreatic adenocarcinoma (PDAC). By QRT-PCR, median mRNA levels of ADM and CRLR were 1.5- and 2.4-fold higher, respectively, in PDAC tissues compared to normal pancreatic tissues. By immunohistochemistry, ADM, CRLR, RAMP1 and RAMP2, but not RAMP3, were expressed in pancreatic cancer cells. ADM serum levels were significantly increased in PDAC patients compared to healthy controls and chronic pancreatitis (CP) patients, with an area under the ROC curve of 0.83 and 0.98, respectively. At a cut-off level of 30.6 ng/ml, the specificity of ADM to differentiate PDAC from controls and CP patients was 85.5 and 83.6%, with a sensitivity of 80 and 100%. All 5 evaluated pancreatic cancer cells lines expressed ADM, CRLR, RAMP1 and RAMP2, whereas RAMP3 was expressed in only 1/5 pancreatic cancer cell lines. ADM was strongly induced by hypoxia and significantly increased invasiveness in 3/5 human pancreatic cancer cells. Blocking of CRLR decreased invasiveness in 4/5 human pancreatic cancer cells. In addition, rADM slightly up-regulated vascular endothelial growth factor secretion in 3/5 cell lines. In conclusion, ADM is induced by hypoxia and over-expressed in PDAC and might therefore serve as a potential tumor marker. Furthermore, ADM increases invasiveness of some pancreatic cancer cells and might influence angiogenesis, suggesting that blocking this pathway might have a therapeutic potential.

Basic transcription factor 3 (BTF3) regulates transcription of tumor-associated genes in pancreatic cancer cells.

Basic transcription factor 3 (BTF3) acts as a transcription factor and modulator of apoptosis, and is differentially expressed in colorectal cancer and glioblastomas. In the present study, the expression of BTF3, as well as its role in apoptosis and gene transcription, was analyzed in pancreatic ductal adenocarcinoma (PDAC). QRT-PCR, immunohistochemistry, immunoblotting, and immunofluorescence analyses were carried out to investigate BTF3 mRNA/protein expression and localization. BTF3 silencing in pancreatic cancer cells was performed using specific siRNA molecules. Functional analyses were carried out using cell growth assays, apoptosis assays, and DNA array analysis. BTF3 and BTF3a exhibited 1.3-fold and 4.6-fold increased median mRNA levels in PDAC tissues, compared to normal pancreatic tissues. BTF3 localized mainly in the cytoplasm and nuclei of tubular complexes and pancreatic cancer cells. Pancreatic cancer cell lines expressed the mRNA and protein of BTF3a (27 kDa) and BTF3b (22 kDa) isoforms. BTF3 silencing using specific siRNA molecules did not influence apoptosis induced by chemotherapy or radiotherapy. In contrast, BTF3 silencing resulted in down-regulation of several cancer-associated genes, including EPHB2, ABL2, HPSE2 and ATM, and up-regulation of KRAG, RRAS2, NFkappa-B, MRVI1, MADCAM1 and others. In conclusion, BTF3 is overexpressed in PDAC, where it acts as a transcriptional regulator rather than a direct modulator of apoptosis.

Tumor-suppressor function of SPARC-like protein 1/Hevin in pancreatic cancer.

SPARC-like protein 1 (SPARCL1), a member of the SPARC family, is downregulated in various tumors. In the present study, the expression and localization of SPARCL1 were analyzed in a wide range of nontumorous and neoplastic pancreatic tissues by quantitative reverse transcription-polymerase chain reaction, laser capture microdissection, microarray analysis, and immunohistochemistry. For functional analysis, proliferation and invasion assays were used in cultured pancreatic cancer cells. Pancreatic ductal adenocarcinoma (PDAC) and other pancreatic neoplasms exhibited increased SPARCL1 mRNA levels compared to those of the normal pancreas. SPARCL1 mRNA levels were low to absent in microdissected and cultured pancreatic cancer cells, and promoter demethylation increased SPARCL1 levels only slightly in three of eight cell lines. SPARCL1 was observed in small capillaries in areas of inflammation/tumor growth and in some islet cells. In PDAC, 15.4% of vessels were SPARCL1-positive. In contrast, the percentage of SPARCL1-positive vessels was higher in chronic pancreatitis and benign and borderline pancreatic tumors. Recombinant SPARCL1 inhibited pancreatic cancer cell invasion and exerted moderate growth-inhibitory effects. In conclusion, SPARCL1 expression in pancreatic tissues is highly correlated with level of vascularity. Its anti-invasive effects and reduced expression in metastasis indicate tumor-suppressor function.

Expression of extracellular matrix metalloproteinase inducer (EMMPRIN/CD147) in pancreatic neoplasm and pancreatic stellate cells.

EMMPRIN (extracellular matrix metalloproteinase inducer, CD147) participates in the progression of various malignancies by stimulating the synthesis of specific matrix metalloproteinases (MMP) from peritumoral fibroblasts. In the present study, the expression and functional role of EMMPRIN was investigated in pancreatic neoplasm. QRT-PCR, immunohistochemistry, immunoblot, and ELISA analyses were used to analyze the expression, localization, and release of EMMRPIN. Silencing of EMMPRIN was performed using siRNA oligonucleotides, and functional consequences were assessed using growth assays, invasion assays, as well as MMP1/MMP2 and VEGF ELISA. EMMPRIN mRNA levels were 2.2-fold increased in pancreatic cancer (n = 52) and 2.0-3.5-fold increased in other pancreatic neoplasm (n = 105), but unchanged in chronic pancreatitis (n = 10) compared to normal pancreatic tissues (n = 9). Strong and predominantly membranous immunostaining was observed in the cancer cells and surrounding stromal cells. EMMPRIN serum levels were also significantly increased in pancreatic cancer patients (n = 44) (4.13 +/- 0.28 ng/ml) with an AUC of 0.97 compared to healthy volunteers (n = 29) (0.95 +/- 0.16 ng/ml; p < 0.0001) and with an AUC of 0.74 compared to chronic pancreatitis patients (n = 20) (2.98 +/- 0.5 ng/ml; p = 0.0021). EMMPRIN silencing did not significantly affect anchorage-dependent or -independent growth of pancreatic cancer cells. In contrast, EMMPRIN silencing in pancreatic stellate cells slightly repressed VEGF and MMP2 levels but strongly increased pro-MMP1 expression under coculture conditions. In conclusion, Increased EMMPRIN expression is present in different pancreatic neoplasm, likely representing a tumor-specific reaction with the potential to modulate the tumor microenvironment rather than a mere reflection of an activated stroma.