Circulating microRNAs: emerging biomarkers for diagnosis and prognosis in patients with gastrointestinal cancers.

To identify novel non-invasive biomarkers for improved detection, risk assessment and prognostic evaluation of cancer, expression profiles of circulating microRNAs are currently under evaluation. Circulating microRNAs are highly promising candidates in this context, as they present some key characteristics for cancer biomarkers: they are tissue-specific with reproducible expression and consistency among individuals from the same species, they are potentially derived directly from the tumour and therefore might correlate with tumour progression and recurrence, and they are bound to proteins or contained in subcellular particles, such as microvesicles or exosomes, making them highly stable and resistant to degradation. The present review highlights the origin of circulating microRNAs, their stability in blood samples, and techniques to isolate exosomal microRNAs, and then addresses the current evidence supporting potential clinical applications of circulating miRNAs for diagnostic and prognostic purposes.

Obstetrics care in Indonesia: Determinants of maternal mortality and stillbirth rates.

PROBLEM: The Indonesian Healthcare Program starting in 2014 enabled access to healthcare delivery for large population groups. Guidance of usage, infrastructure and healthcare process development were the most challenging tasks during the implementation period. Due to the high social impact obstetric care and related quality assurance require evidence-based developmental strategies. This study aims for analysis of outcome and maternal health care utilization, as well as differences related to demographic and economic subgroups. METHODS: For univariate group comparison ANOVA method was applied and combined with Scheffé procedure and Bonferoni correction for post-hoc tests. Meanwhile, multivariate approaches through regression analysis based on insurance reimbursement data antenatal, perinatal and postnatal care were performed at the province level. Maternal mortality (MMR) and stillbirth rates were used for outcome. Demographic characteristics, availability of obstetricians (SPOG), midwifes and healthcare infrastructure were included for their determinants. RESULTS: Specialized hospital facilities (type A/B) for advanced care covered a large part of uncomplicated cases (~35%). Differences between insurance membership groups (poor, non-poor) were not seen. Availability of human resources (SPOG, midwifes) (R2 = 0.728; p<0.001) and rural setting (R2 = 0.288; p = 0.001) are correlated with reduced insufficient referral. Their presence within provinces was related to lower occurrence of complicated cases (R2 = 0.294; p = 0.001). However, higher SPOG rates within provinces were also related to high C-section rates (p<0.001). MMR and stillbirth rates can be predicted by availability of human resources and C-section rates explaining 49.0% of variance. CONCLUSIONS: Improvement of perinatal outcome should focus on sufficient referral processes, availability of SPOG in provinces dominated by rural/remote demography and avoidance of overtreatment by high C-section rates. It is very important to regulate the education of obstetricians and gynecologists in Indonesia as well as distribution arrangements regarding to solve the problems with pregnancy complications in remote and rural areas.

Epigenetic regulation and role of metastasis suppressor genes in pancreatic ductal adenocarcinoma.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is distinguished by rapid dissemination. Thus, genetic and/or epigenetic deregulation of metastasis suppressor genes (MSG) is a likely event during early pancreatic carcinogenesis and a potential diagnostic marker for the disease. We investigated 9 known MSGs for their role in the dissemination of PDAC and examined their promoters for methylation and its use in PDAC detection. METHODS: MRNA expression of 9 MSGs was determined in 18 PDAC cell lines by quantitative RT-PCR and promoter methylation was analyzed by Methylation Specific PCR and validated by Bisulfite Sequencing PCR. These data were compared to the cell lines' in vivo metastatic and invasive potential that had been previously established. Statistical analysis was performed with SPSS 20 using 2-tailed Spearman's correlation with P < 0.05 being considered significant. RESULTS: Complete downregulation of MSG-mRNA expression in PDAC cell lines vs. normal pancreatic RNA occurred in only 1 of 9 investigated genes. 3 MSGs (CDH1, TIMP3 and KiSS-1) were significantly methylated. Methylation only correlated to loss of mRNA expression in CDH1 (P < 0.05). Bisulfite Sequencing PCR showed distinct methylation patterns, termed constant and variable methylation, which could distinguish methylation-regulated from non methylation-regulated genes. Higher MSG mRNA-expression did not correlate to less aggressive PDAC-phenotypes (P > 0.14). CONCLUSIONS: Genes with metastasis suppressing functions in other tumor entities did not show evidence of assuming the same role in PDAC. Inactivation of MSGs by promoter methylation was an infrequent event and unsuitable as a diagnostic marker of PDAC. A distinct methylation pattern was identified, that resulted in reduced mRNA expression in all cases. Thus, constant methylation patterns could predict regulatory significance of a promoter's methylation prior to expression analysis and hence present an additional tool during target gene selection.

Mimicry of human sepsis in a rat model--prospects and limitations.

BACKGROUND: Sepsis and systemic inflammatory response syndrome (SIRS) continue to represent critical conditions with persistently high mortality and continue to need experimental and clinical research. We developed a rat model of gram-positive and gram-negative SIRS/sepsis with in vivo visualization of the pulmonary microcirculation to evaluate the optimal dosage and application path for SIRS/sepsis-inducing agents. METHODS: Male Sprague-Dawley rats (n = 8 per group) were assigned to control, lipopolysaccharide (LPS), alphatoxin, or living Staphylococcus aureus (strain 68/50) groups. SIRS/sepsis was induced by intraperitoneal injection of the differing agents. The onset of SIRS was determined through human sepsis parameters and fluorescence video microscopy-based measurement of platelet and leukocyte velocity within the pulmonary vascular system (injection of 5 × 10(6) calcein AM-labeled nonactivated platelets; leukocytes labeled in vivo by rhodamine). RESULTS: The optimal dosage to induce SIRS was 30 mg/250 g body weight for LPS (bolus injection) and 60 µg/250 g body weight for alphatoxin (2 h continuous perfusion). Sepsis was not achieved by injection of living S. aureus. The onset of SIRS was seen after 2-5 h for LPS and after 2-4 h for alphatoxin after intraperitoneal administration with a significantly increased heart rate, breathing rate, and body temperature (P < 0.05) and significantly decreased cell velocity (P < 0.05). CONCLUSION: Our study represents an effective approach for a gram-negative (LPS) and gram-positive (alphatoxin) SIRS model to mimic human sepsis. Human sepsis-based criteria were used to define SIRS in our rats to achieve an optimal analogy for the human system. In our model, higher dosages were needed for SIRS induction than have been previously reported. The resulting, considerable heterogeneity of current SIRS-inducing models suggests that additional studies in this field are required to define standard procedures.

Blended learning in surgery using the Inmedea Simulator.

BACKGROUND: Recently, medical education in surgery has experienced several modifications. We have implemented a blended learning module in our teaching curriculum to evaluate its effectiveness, applicability, and acceptance in surgical education. METHODS: In this prospective study, the traditional face-to-face learning of our teaching curriculum for fourth-year medical students (n = 116) was augmented by the Inmedea Simulator, a web-based E-learning system, with six virtual patient cases. Student results were documented by the system and learning success was determined by comparing patient cases with comparable diseases (second and sixth case). The acceptance among the students was evaluated with a questionnaire. RESULTS: After using the Inmedea Simulator, correct diagnoses were found significantly (P < 0.05) more often, while an incomplete diagnostic was seen significantly (P < 0.05) less often. Significant overall improvement (P < 0.05) was seen in sixth case (62.3 ± 5.6 %) vs. second case (53.9 ± 5.6 %). The questionnaire revealed that our students enjoyed the surgical seminar (score 2.1 ± 1.5) and preferred blended learning (score 2.5 ± 1.2) to conventional teaching. CONCLUSION: The blended learning approach using the Inmedea Simulator was highly appreciated by our medical students and resulted in a significant learning success. Blended learning appears to be a suitable tool to complement traditional teaching in surgery.

Economic Perspective of Cancer Care and Its Consequences for Vulnerable Groups.

Within healthcare systems in all countries, vulnerable groups of patients can be identified and are characterized by the reduced utilization of available healthcare. Many different reasons can be attributed to this observation, summarized as implementation barriers involving acceptance, accessibility, affordability, acceptability and quality of care. For many patients, cancer care is specifically associated with the occurrence of vulnerability due to the complex disease, very different target groups and delivery situations (from prevention to palliative care) as well as cost-intensive care. Sociodemographic factors, such as educational level, rural/remote location and income, are known determinants for these vulnerable groups. However, different forms of financial burdens likely influence this vulnerability in cancer care delivery in a distinct manner. In a narrative review, these socioeconomic challenges are summarized regarding their occurrence and consequences to current cancer care. Overall, besides direct costs such as for treatment, many facets of indirect costs including survivorship costs for the cancer patients and their social environment need to be considered regarding the impact on vulnerability, treatment compliance and abundance. In addition, individual cancer-related financial burden might also affect the society due to the loss of productivity and workforce availability. Healthcare providers are requested to address this vulnerability during the treatment of cancer patients.

A pyramid model to describe changing decision making under high uncertainty during the COVID-19 pandemic.

The COVID-19 pandemic put healthcare systems, hospitals and medical personal under great pressure. Based on observations in Germany, we theorise a general model of rapid decision-making that makes sense of the growing complexity, risks and impact of missing evidence. While adapting decision-making algorithms, management, physicians, nurses and other healthcare professionals had to move into uncharted territory while addressing practical challenges and resolving normative (legal and ethical) conflicts. During the pandemic, this resulted in decisional uncertainties for healthcare professionals. We propose an idealised risk-based model that anticipates these shifts in decision-making procedures and underlying value frameworks. The double pyramid model visualises foreseeable procedural adaptations. This does not only help practitioners to secure operational continuity in a crisis but also contributes to improving the conceptual underpinnings of the resilience of healthcare during the next pandemic or similar future crises situations.

Population-based study on coverage and healthcare processes for cancer during implementation of national healthcare insurance in Indonesia.

Background: A national healthcare insurance has been implemented in Indonesia since 2014. Although cancer care currently represents a smaller part of the healthcare support, the demographic development will lead to a rapid growth of the population within age groups at cancer risk. This requires strategic and developmental planning of cancer care resources. Based on data of the national healthcare insurance, current cancer care processes and their determinants were evaluated. Methods: Nationwide reimbursement data as well as demographic, economic and healthcare infrastructure data were used for the study. Poor and underserved population was stratified according to the national classification system. Availability of healthcare resources was evaluated at provincial level. Cancer care usage was analysed applying descriptive and multivariate statistical approaches (regression, cluster analysis, tree classification). Findings: Cancer care was provided in primary care (PHC) for 2.6/1000 and advanced care (AHC) for 4.8/1000 participants within the family-based membership structure. Regression analysis revealed human resource availability in rural/remote areas a determinant for cancer PHC. Cancer care in AHC was determined by PHC provided by general practitioners (GP), availability of AHC infrastructure (Class A & B hospital beds) and treatment migration between provinces. Tree classification confirmed predominant roles of GP, AHC infrastructure and referral between cancer care provider levels. Interpretation: Cancer care will gain much higher importance for the Indonesian healthcare system within the next decade. Infrastructure, human resources, and process development should avoid rising overload of cancer care delivery by targeting reduction of treatment migration (availability of GPs in rural/remote provinces), improvement of referral systems (effective clinical selection processes and back-referral) and AHC cancer care structures (regional distribution of Class A & B hospitals). Funding: This project was supported by grants from Centre for Research, Publication, and Community Development Muhammadiyah University of Yogyakarta (SW, ID), and data provision by BPJS Indonesia.

Decision Conflicts in Clinical Care during COVID-19: A Multi-Perspective Inquiry.

Background: The early COVID-19-pandemic was characterized by changes in decision making, decision-relevant value systems and the related perception of decisional uncertainties and conflicts resulting in decisional burden and stress. The vulnerability of clinical care professionals to these decisional dilemmas has not been characterized yet. Methods: A cross-sectional questionnaire study (540 patients, 322 physicians and 369 nurses in 11 institutions throughout Germany) was carried out. The inclusion criterion was active involvement in clinical treatment or decision making in oncology or psychiatry during the first year of COVID-19. The questionnaires covered five decision dimensions (conflicts and uncertainty, resources, risk perception, perception of consequences for clinical processes, and the perception of consequences for patients). Data analysis was performed using ANOVA, Pearson rank correlations, and the Chi²-test, and for inferential analysis, nominal logistic regression and tree classification were conducted. Results: Professionals reported changes in clinical management (27.5%) and a higher workload (29.2%), resulting in decisional uncertainty (19.2%) and decisional conflicts (22.7%), with significant differences between professional groups (p < 0.005), including anxiety, depression, loneliness and stress in professional subgroups (p < 0.001). Nominal regression analysis targeting "Decisional Uncertainty" provided a highly significant prediction model (LQ p < 0.001) containing eight variables, and the analysis for "Decisional Conflicts" included six items. The classification rates were 64.4% and 92.7%, respectively. Tree analysis confirmed three levels of determinants. Conclusions: Decisional uncertainty and conflicts during the COVID-19 pandemic were independent of the actual pandemic load. Vulnerable professional groups for the perception of a high number of decisional dilemmas were characterized by individual perception and the psychological framework. Coping and management strategies should target vulnerability, enable the handling of the individual perception of decisional dilemmas and ensure information availability and specific support for younger professionals.

Differences in Stakeholders' Perception of the Impact of COVID-19 on Clinical Care and Decision-Making.

Background: Pandemics are related to changes in clinical management. Factors that are associated with individual perceptions of related risks and decision-making processes focused on prevention and vaccination, but perceptions of other healthcare consequences are less investigated. Different perceptions of patients, nurses, and physicians on consequences regarding clinical management, decisional criteria, and burden were compared. Study Design: Cross-sectional OnCoVID questionnaire studies. Methods: Data that involved 1231 patients, physicians, and nurses from 11 German institutions that were actively involved in clinical treatment or decision-making in oncology or psychiatry were collected. Multivariate statistical approaches were used to analyze the stakeholder comparisons. Results: A total of 29.2% of professionals reported extensive changes in workload. Professionals in psychiatry returned severe impact of pandemic on all major aspects of their clinical care, but less changes were reported in oncology (p < 0.001). Both patient groups reported much lower recognition of treatment modifications and consequences for their own care. Decisional and pandemic burden was intensively attributed from professionals towards patients, but less in the opposite direction. Conclusions: All of the groups share concerns about the impact of the COVID-19 pandemic on healthcare management and clinical processes, but to very different extent. The perception of changes is dissociated in projection towards other stakeholders. Specific awareness should avoid the dissociated impact perception between patients and professionals potentially resulting in impaired shared decision-making.

Epigenetic markers for chemosensitivity and chemoresistance in pancreatic cancer--a review.

Adjuvant first-line gemcitabine monochemotherapy presents a standard treatment for patients with advanced pancreatic adenocarcinoma and improves overall survival in chemosensitive patients. Nonetheless, 6-month progression-free survival remains below 15%, despite interdisciplinary approaches. The success of gemcitabine treatment is disappointing and-in the absence of reliable tumor markers--challenging to quantify. Epigenetic alterations have been recently identified to take on important roles in cancer development and possibly cancer treatment. In this context, microRNAs are becoming increasingly acknowledged as useful biomarkers for classifying cancers and providing information on their chemo- and radiosensitivity. This review illustrates the potential of genetic and epigenetic markers in the prediction of chemosensitivity in pancreatic cancer patients and in the monitoring of their response rates to adjuvant therapy.

MiRNAs and their association with locoregional staging and survival following surgery for esophageal carcinoma.

BACKGROUND: Prognostic and staging information for esophageal cancer impacts clinical decision making. miRNAs, a newly discovered class of biomarkers and their expression might add additional information relevant to this. In this study we evaluated the expression of selected miRNAs and their relationship to tumor stage and survival in patients with locally advanced tumors following esophagectomy. MATERIALS AND METHODS: A total of 43 individuals undergoing esophagectomy (without neoadjuvant therapy) for locally advanced but not metastatic (pT2/3; pN0/1) disease (22 adenocarcinoma [EAC], 21 squamous cell carcinoma [SCC]) were included in this study. Perioperative clinical and survival data were collected and managed on a database. The expression of miR-21, miR-106a, miR-148a, miR-205 in formalin-fixed paraffin-embedded specimens was evaluated by TaqMan qPCR assays. Expression was compared with clinicopathological features of the cancers and outcome. RESULTS: In EAC, miR-148a expression levels were inversely associated with cancer differentiation. miR-21 expression levels were higher in SCC if distant lymph node metastases were present. miR-148a levels were lower when EAC was more proximally located, and miR-21 levels were lower when SCC was more proximal. miR-106a and miR-148a were lower in patients with SCC who developed recurrent disease or had a tumor-related death. CONCLUSIONS: In patients with locally advanced esophageal squamous cell carcinoma, but not adenocarcinoma, alterations in the expression of miR-21 correlate with tumor location and lymph node status. Furthermore, miR-106a and miR-148a expression correlates with disease recurrence and tumor-related mortality. miRNA markers might inform the initial assessment of these patients, and predict those at higher risk of postsurgical recurrence.

Analyzing miRNAs in ductal adenocarcinomas of the pancreas.

BACKGROUND: MicroRNAs (miRNAs) have gained attention as an epigenetic component involved in the development of pancreatic ductal adenocarcinoma (PDAC). Several methods for miRNA profiling are in common use, but the validity of these methods is not defined. The aim of this study was to define the optimal method for miRNA detection in PDAC. METHODS: miRNA expression was determined using different and partially redundant methods (miRNA microarray, TaqMan low density array (TLDA), single tube quantitative RT-PCR). The data from different methods were statistically evaluated and tested for intermethodic consistency and reliability of the results. Finally, the miRNA expression status and the cell lines' ability to metastasize were correlated. RESULTS: Comparing low and high metastatic cells, miRNA-microarrays identified fewer differentially expressed and only upregulated miRNAs (n=27; 27 up-regulated) compared with TLDAs (n=54; 19 up- and 35 down-regulated). Evaluating miRNAs that target tumor suppressor genes, expression of all single tube quantitative real-time reverse transcriptase PCR (qRT-PCR) validated miRNAs was detected to be significantly altered in TLDA analysis (100%). MiRNA microarrays detected only 25% of qRT-PCR validated miRNAs. Furthermore, results from TLDA analysis correlated well with data from qRT-PCR and presented ΔΔCt values from 3.5±1.86 (range 0.8-5.62) compared with 3.74±1.86 (range 0.78-5.95) in qRT-PCR. CONCLUSION: Notable differences comparing data obtained from different screening methods were found. While TLDA and qRT-PCR correlated well in quantity and quality of the measured miRNAs, several tumor suppressor gene targeting and down-regulated miRNAs were not detected by miRNA-microarrays. This heterogeneity shows that care must be exercised when comparing results from different methods in PDAC.

Role of tumor microenvironment on gene expression in pancreatic cancer tumor models.

OBJECTIVES: The microenvironment is known to be a relevant factor of influence on tumor growth and metastasis in pancreatic ductal adenocarcinoma (PDAC). To determine the influence of the microenvironment on changes in gene expression, we analyzed gene expression in different PDAC tissues. METHODS: Four human PDAC cell lines were introduced into a murine PDAC model with two insertion techniques: injection and implantation. Gene expression profiles of the cell lines growing in vitro and in vivo (ectopically and orthotopically) were established by microarray and validated by RT-PCR. RESULTS: Significant differences were found in the gene expression profiles of the in vitro versus in vivo tissues (P < 0.05), while no differences were found between the in vivo tissues. Analyzing the orthotopic tumors derived from the injection and implantation methods, similar gene expression patterns with 0%-18% significantly differentially expressed genes between tumors of the two different methods were observed (analysis of variance [ANOVA]; P < 0.0001). CONCLUSIONS: Gene expression from cell lines growing in vitro differed from the expression patterns of the same cells growing in vivo, while the localization of the growing tumor cells did not significantly alter gene expression. These data demonstrate that the implantation and injection techniques used in this study yield similar results and may be compared with each other.

Availability and Accessibility of Primary Care for the Remote, Rural, and Poor Population of Indonesia.

Background: Adopting Universal Health Coverage for implementation of a national health insurance system [Jaminan Kesehatan Nasional (JKN)/Badan Penyelenggara Jaminan Sosial or the Indonesian National Social Health Insurance Scheme (BPJS)] targets the 255 million population of Indonesia. The availability, accessibility, and acceptance of healthcare services are the most important challenges during implementation. Referral behavior and the utilization of primary care structures for underserved (rural/remote regions) populations are key guiding elements. In this study, we provided the first assessment of BPJS implementation and its resulting implications for healthcare delivery based on the entire insurance dataset for the initial period of implementation, specifically focusing on poor and remote populations. Methods: Demographic, economic, and healthcare infrastructure information was obtained from public resources. Data about the JKN membership structure, performance information, and reimbursement were provided by the BPJS national head office. For analysis, an ANOVA was used to compare reimbursement indexes for primary healthcare (PHC) and advanced healthcare (AHC). The usage of primary care resources was analyzed by comparing clustered provinces and utilization indices differentiating poor [Penerima Bantuan Iur (PBI) membership] and non-poor populations (non-PBI). Factorial and canonical discrimination analyses were applied to identify the determinants of PHC structures. Results: Remote regions cover 27.8% of districts/municipalities. The distribution of the poor population and PBI members were highly correlated (r2 > 0.8; p < 0.001). Three clusters of provinces [remote high-poor (N = 13), remote low-poor (N = 15), non-remote (N = 5)] were identified. A discrimination analysis enabled the >82% correct cluster classification of infrastructure and human resources of health (HRH)-related factors. Standardized HRH (nurses and general practitioners [GP]) availability showed significant differences between clusters (p < 0.01), whereas the availability of hospital beds was weakly correlated. The usage of PHC was ~2-fold of AHC, while non-PBI members utilized AHC 4- to 5-fold more frequently than PBI members. Referral indices (r2 = 0.94; p < 0.001) for PBI, non-PBI, and AHC utilization rates (r2 = 0.53; p < 0.001) were highly correlated. Conclusion: Human resources of health availability were intensively related to the extent of the remote population but not the numbers of the poor population. The access points of PHC were mainly used by the poor population and in remote regions, whereas other population groups (non-PBI and non-Remote) preferred direct access to AHC. Guiding referral and the utilization of primary care will be key success factors for the effective and efficient usage of available healthcare infrastructures and the achievement of universal health coverage in Indonesia. The short-term development of JKN was recommended, with a focus on guiding referral behavior, especially in remote regions and for non-PBI members.

EP300--a miRNA-regulated metastasis suppressor gene in ductal adenocarcinomas of the pancreas.

Genetic and epigenetic alterations during development of pancreatic ductal adenocarcinomas (PDACs) are well known. This study investigates genetic and epigenetic data together with tumor biology to find specific alterations responsible for metastasis formation. Using 16 human PDAC cell lines in a murine orthotopic PDAC model, local infiltration and metastatic spread were assessed by standardized dissemination scores. The cell lines were further classified into 3 hierarchical groups according to their metastatic potential. Their mRNA and microRNA (miRNA) expression was profiled via mRNA-microarray as well as Taqman Low Density Array, and validated by single quantitative RT-PCR and Western blotting. In the highly metastatic group, a significant induction of EP300 targeting miRNAs miR-194 (fold change: 26.88), miR-200b (fold change: 61.65), miR-200c (fold change: 19.44) and miR-429 (fold change: 21.67) (p < 0.05) was detected. Corresponding to this, decreased expression of EP300 mRNA (p < 0.0001) and protein (p < 0.05) were detected in the highly metastatic PDAC cell lines with liver metastases compared to the nonmetastatic or marginally metastatic cell lines, while no correlation with local tumor growth was found. In conclusion, epigenetic alterations with upregulated EP300 targeting miRNAs miR-194, miR-200b, miR-200c and miR-429 are related to reduced EP300 mRNA and protein in PDAC. These results demonstrate that miRNAs might be able to modulate the expression of metastasis-specific suppressor genes and metastatic behavior in PDAC, suggesting diagnostic and therapeutic opportunities for EP300 and its targeting miRNAs in PDAC.

In vivo tumor cell adhesion in the pulmonary microvasculature is exclusively mediated by tumor cell--endothelial cell interaction.

BACKGROUND: Metastasis formation is the leading cause of death among colon cancer patients. We established a new in-situ model of in vivo microscopy of the lung to analyse initiating events of metastatic tumor cell adhesion within this typical metastatic target of colon cancer. METHODS: Anaesthetized CD rats were mechanically ventilated and 106 human HT-29LMM and T84 colon cancer cells were injected intracardially as single cell suspensions. Quantitative in vivo microscopy of the lung was performed in 10 minute intervals for a total of 40 minutes beginning with the time of injection. RESULTS: After vehicle treatment of HT-29LMM controls 15.2 +/- 5.3; 14.2 +/- 7.5; 11.4 +/- 5.5; and 15.4 +/- 6.5 cells/20 microscopic fields were found adherent within the pulmonary microvasculature in each 10 minute interval. Similar numbers were found after injection of the lung metastasis derived T84 cell line and after treatment of HT-29LMM with unspecific mouse control-IgG. Subsequently, HT-29LMM cells were treated with function blocking antibodies against beta1-, beta4-, and alphav-integrins wich also did not impair tumor cell adhesion in the lung. In contrast, after hydrolization of sialylated glycoproteins on the cells' surface by neuraminidase, we observed impairment of tumor cell adhesion by more than 50% (p < 0.05). The same degree of impairment was achieved by inhibition of P- and L-selectins via animal treatment with fucoidan (p < 0.05) and also by inhibition of the Thomson-Friedenreich (TF)-antigen (p < 0.05). CONCLUSIONS: These results demonstrate that the initial colon cancer cell adhesion in the capillaries of the lung is predominantly mediated by tumor cell - endothelial cell interactions, possibly supported by platelets. In contrast to reports of earlier studies that metastatic tumor cell adhesion occurs through integrin mediated binding of extracellular matrix proteins in liver, in the lung, the continuously lined endothelium appears to be specifically targeted by circulating tumor cells.

SERPINB5 and AKAP12 - expression and promoter methylation of metastasis suppressor genes in pancreatic ductal adenocarcinoma.

BACKGROUND: Early metastasis and infiltration are survival limiting characteristics of pancreatic ductal adenocarcinoma (PDAC). Thus, PDAC is likely to harbor alterations in metastasis suppressor genes that may provide novel diagnostic and therapeutic opportunities. This study investigates a panel of metastasis suppressor genes in correlation to PDAC phenotype and examines promoter methylation for regulatory influence on metastasis suppressor gene expression and for its potential as a diagnostic tool. METHODS: Metastatic and invasive potential of 16 PDAC cell lines were quantified in an orthotopic mouse model and mRNA expression of 11 metastasis suppressor genes determined by quantitative RT-PCR. Analysis for promoter methylation was performed using methylation specific PCR and bisulfite sequencing PCR. Protein expression was determined by Western blot. RESULTS: In general, higher metastasis suppressor gene mRNA expression was not consistent with less aggressive phenotypes of PDAC. Instead, mRNA overexpression of several metastasis suppressor genes was found in PDAC cell lines vs. normal pancreatic RNA. Of the investigated metastasis suppressor genes, only higher AKAP12 mRNA expression was correlated with decreased metastasis (P < 0.05) and invasion scores (P < 0.01) while higher SERPINB5 mRNA expression was correlated with increased metastasis scores (P < 0.05). Both genes' promoters showed methylation, but only increased SERPINB5 methylation was associated with loss of mRNA and protein expression (P < 0.05). SERPINB5 methylation was also directly correlated to decreased metastasis scores (P < 0.05). CONCLUSIONS: AKAP12 mRNA expression was correlated to attenuated invasive and metastatic potential and may be associated with less aggressive phenotypes of PDAC while no such evidence was obtained for the remaining metastasis suppressor genes. Increased SERPINB5 mRNA expression was correlated to increased metastasis and mRNA expression was regulated by methylation. Thus, SERPINB5 methylation was directly correlated to metastasis scores and may provide a diagnostic tool for PDAC.

Prognostic impact of underlying liver fibrosis and cirrhosis after curative resection of hepatocellular carcinoma.

BACKGROUND: In the case of hepatocellular carcinoma (HCC), underlying liver pathology may not only determine the feasibility of surgery but may also affect the postsurgical outcome. We report our experience after curative liver resection for HCC in patients with normal liver, liver fibrosis, and liver cirrhosis. METHODS: A total of 72 patients after liver resection with curative intention were analyzed. Histopathologic findings of tumor-unaffected liver tissue were used for retrospective classification: group A (normal liver); group B (liver fibrosis); group C (liver cirrhosis). The groups were compared for differences in short-term surgical results, total survival, and recurrence-free survival. RESULTS: The rate of major complications was 34.7% and did not significantly differ among groups. The overall perioperative mortality rate was 9.7%, with one patient dying in group A and three patients dying in each of the other two groups. Including perioperative mortality, the median overall survival for the whole group was 37.3 months (95% confidence interval 29.3-45.2 months). The respective 1-, 2-, and 5-year survival rates for group A (n = 21) were 86%, 71%, and 50% and for group C (n = 24) 62%, 50%, and 17%. The overall survival of group B (n = 27) was intermediate (log-rank, P = 0.032). The respective recurrence-free survival rates were 76%, 42%, and 20% for group A and 39%, 13%, and 4% for group C, with group B being intermediate (log-rank, P = 0.016). CONCLUSIONS: Our data demonstrate that liver resection in the presence of compensated liver cirrhosis is feasible but associated with a significantly impaired prognosis for overall and recurrence-free survival. The management of cirrhotic patients with compensated liver function and HCC therefore also requires the opportunity for transplantation.

Involvement of CD40 targeting miR-224 and miR-486 on the progression of pancreatic ductal adenocarcinomas.

BACKGROUND: Genetic and epigenetic alterations during development of pancreatic ductal adenocarcinomas (PDAC) are well known. Genetic and epigenetic data were correlated with tumor biology to find specific alterations responsible for invasion and metastasis in pancreatic ductal adenocarcinomas. METHODS: A total of 16 human PDAC cell lines were used in murine orthotopic PDAC models. By means of standardized dissemination scores, local invasion and metastatic spread were assessed. mRNA and microRNA expression were studied by microarray and TaqMan low-density array. Quantitative real-time-polymerase chain reaction and flow cytometry were used for expression validation. RESULTS: CD40 was detected as a relevant target gene for differentially expressed miRNAs observed in highly invasive and metastatic PDAC only. A significant overexpression (P < .05) of CD40-related miRNAs miR-224 and miR-486 was detected in highly invasive and metastatic PDAC, whereas CD40 mRNA expression was not significantly altered. Instead, CD40 protein expression at cell surfaces of these highly invasive and metastatic PDAC was significantly reduced (P < .01). CONCLUSIONS: Epigenetic alterations with upregulated CD40-targeting miR-224 and miR-486 are related to downregulated CD40 protein expression at cell surfaces in highly invasive and metastatic PDAC. Thus, miRNA-regulated CD40 expression seems to play an important role in progression of PDAC. These data suggest a diagnostic and therapeutic potential for CD40 and/or its targeting miRNAs in PDAC.