3D Visualization, Skeletonization and Branching Analysis of Blood Vessels in Angiogenesis.

Angiogenesis is the process of new blood vessels growing from existing vasculature. Visualizing them as a three-dimensional (3D) model is a challenging, yet relevant, task as it would be of great help to researchers, pathologists, and medical doctors. A branching analysis on the 3D model would further facilitate research and diagnostic purposes. In this paper, a pipeline of vision algorithms is elaborated to visualize and analyze blood vessels in 3D from formalin-fixed paraffin-embedded (FFPE) granulation tissue sections with two different staining methods. First, a U-net neural network is used to segment blood vessels from the tissues. Second, image registration is used to align the consecutive images. Coarse registration using an image-intensity optimization technique, followed by finetuning using a neural network based on Spatial Transformers, results in an excellent alignment of images. Lastly, the corresponding segmented masks depicting the blood vessels are aligned and interpolated using the results of the image registration, resulting in a visualized 3D model. Additionally, a skeletonization algorithm is used to analyze the branching characteristics of the 3D vascular model. In summary, computer vision and deep learning is used to reconstruct, visualize and analyze a 3D vascular model from a set of parallel tissue samples. Our technique opens innovative perspectives in the pathophysiological understanding of vascular morphogenesis under different pathophysiological conditions and its potential diagnostic role.

Mesenchymal Stromal Cell Therapy in Novel Porcine Model of Diffuse Liver Damage Induced by Repeated Biliary Obstruction.

In liver surgery, biliary obstruction can lead to secondary biliary cirrhosis, a life-threatening disease with liver transplantation as the only curative treatment option. Mesenchymal stromal cells (MSC) have been shown to improve liver function in both acute and chronic liver disease models. This study evaluated the effect of allogenic MSC transplantation in a large animal model of repeated biliary obstruction followed by partial hepatectomy. MSC transplantation supported the growth of regenerated liver tissue after 14 days (MSC group, n = 10: from 1087 ± 108 (0 h) to 1243 ± 92 mL (14 days); control group, n = 11: from 1080 ± 95 (0 h) to 1100 ± 105 mL (14 days), p = 0.016), with a lower volume fraction of hepatocytes in regenerated liver tissue compared to resected liver tissue (59.5 ± 10.2% vs. 70.2 ± 5.6%, p < 0.05). Volume fraction of connective tissue, blood vessels and bile vessels in regenerated liver tissue, serum levels of liver enzymes (AST, ALT, ALP and GGT) and liver metabolites (albumin, bilirubin, urea and creatinine), as well as plasma levels of IL-6, IL-8, TNF-α and TGF-ß, were not affected by MSC transplantation. In our novel, large animal (pig) model of repeated biliary obstruction followed by partial hepatectomy, MSC transplantation promoted growth of liver tissue without any effect on liver function. This study underscores the importance of translating results between small and large animal models as well as the careful translation of results from animal model into human medicine.

Role of blood biomarkers in spontaneous intracerebral hemorrhage - a prospectively studied cohort of patients.

The study of blood biomarkers can offer new possibilities in diagnostics, prognostication, determination of etiology, and management of spontaneous intracerebral hemorrhage. The aim of our study was to assess the relationship between a panel of selected blood biomarkers and clinical and radiodiagnostic parameters in patients with spontaneous intracerebral hemorrhage. Primarily, the aim was to find a prognostic biomarker which could help in deciding on the optimal categorization of treatment. A total of 70 patients were prospectively included in this study. As shown by our findings, higher levels of S100B protein are associated with larger hematoma volume. They predict hematoma progression and an unfavorable outcome. One other positive correlation was found between hematoma volume and interleukin 6, interleukin 10 and blood glucose. Lower levels of matrix metalloproteinase 9 are an independent prognostic factor for hematoma progression in patients with spontaneous intracerebral hemorrhage.

Evaluation of Serum Cytokeratines, Thymidine Kinase, and Growth Factors as Cancer Biomarkers in Colorectal Cancer.

BACKGROUND/AIM: Classical serum cancer biomarkers, such as carcinoembryonic antigen (CEA) and cancer antigen 19-9 (CA 19-9), remain important tools in colorectal cancer (CRC) management for disease follow up. However, their sensitivity and specificity are low for diagnostic and prognostic evaluation. The aim of this study was to evaluate the potential of biomarkers reflecting biological activity of tumors - tissue polypeptide specific antigen (TPS), cytokeratin fragment 19 (CYFRA 21-1), thymidine kinase (TK), insulin-like growth factor 1 (IGF-1) and insulin-like growth factor binding protein 3 (IGF-BP3) - together with the CEA and CA 19-9 in CRC diagnosis and prognosis. PATIENTS AND METHODS: This is a retrospective study including 148 CRC patients and 68 age-matched healthy subjects. Serum biomarkers were measured in pre-operative serum samples using immunoanalytical methods. The end-point for the diagnostic evaluation was the area under the receiving operating characteristic curve (AUC ROC) of the biomarkers. The end-point for the prognostic evaluation was overall survival. RESULTS: Serum levels of CEA, CA 19-9, TPS, and TK were significantly increased in CRC early-stage patients compared with healthy controls. Each of the studied biomarkers had AUC between 0.6 and 0.7. Analysis of survival demonstrated that the patients with CEA, CA 19-9, cytokeratin, and TK above optimal cut offs had significantly shorter survival. A multivariate analysis performed on all the study biomarkers resulted in the selection of CYFRA 21-1 as the best performing biomarker with hazard ratio 10.413. CONCLUSION: The combination of cytokeratins and thymidine kinase with classical cancer biomarkers enables the prediction of tumor aggressiveness and long-term prognosis.

Establishing Biobanking in Medical Curricula-The Education Program "Precision Medicine International" (eduBRoTHER).

Biobanking is becoming increasingly important as a key tool for precision medicine, but neither biobanking nor precision medicine itself have generally been integrated in medical curricula. However, most medical students will encounter these topics in their future careers as physicians or researchers. The European Union (EU)-funded project eduBRoTHER aims to close this gap of professional input. Since the academic year 2020/21, students at the Faculty of Medicine of Pilsen-Charles University and the Faculty of Medicine of the University of Regensburg have been offered an innovative core elective subject that focuses on biobanking and precision medicine issues, using the concept of blended learning.

Plasma microRNA Levels Combined with CEA and CA19-9 in the Follow-Up of Colorectal Cancer Patients.

Colorectal cancer (CRC) ranks among the most common cancers worldwide. Surgical removal remains the best strategy for treatment of resectable tumors. An important part of caring for patients after surgery is monitoring for early detection of a possible relapse of the disease. Efforts are being made to improve the sensitivity and specificity of routinely used carcinoembryonic antigen (CEA) with the use of additional biomarkers such as microRNAs. The aim of our study was to evaluate the prognostic potential of microRNAs and their use as markers of disease recurrence. The quantitative estimation of CEA, CA19-9, and 22 selected microRNAs (TaqMan Advanced miRNA Assays) was performed in 85 paired (preoperative and postoperative) blood plasma samples of CRC patients and in samples taken during the follow-up period. We have revealed a statistically significant decrease in plasma levels for miR-20a, miR-23a, miR-210, and miR-223a (p = 0.0093, p = 0.0013, p = 0.0392, and p = 0.0214, respectively) after surgical removal of the tumor tissue. A statistically significant relation to prognosis (overall survival; OS) was recorded for preoperative plasma levels of miR-20a, miR-21, and miR-23a (p = 0.0236, p = 0.0316, and p =0.0271, respectively) in a subgroup of patients who underwent palliative surgery. The best discrimination between patients with favorable and unfavorable outcomes was achieved by a combination of CEA, CA19-9 with miR-21, miR-20a, and miR-23a (p < 0.0001). The use of these microRNAs for early disease recurrence detection was affected by a low specificity in comparison with CEA and CA19-9. CEA and CA19-9 had high specificity but low sensitivity. Our results show the benefit of combining currently used standard biomarkers and microRNAs for precise prognosis estimation.

Blood biomarker panel recommended for personalized prediction, prognosis, and prevention of complications associated with abdominal aortic aneurysm.

The aim of the study was to evaluate the ability of following biomarkers as diagnostic tools and risk predictors of AAA: C-reactive protein, interleukin-6, pentraxin-3, galectin-3, procollagen type III N-terminal peptide, C-terminal telopeptide of type I collagen, high-sensitive troponin I, and brain natriuretic peptide. Seventy-two patients with an AAA and 100 healthy individuals were enrolled into the study. We assessed individual biomarker performance and correlation between the AAA diameter and biomarker levels, and also, a multivariate logistic regression was used to design a possible predictive model of AAA growth and rupture risk. We identified following four parameters with the highest potential to find a useful place in AAA diagnostics: galectin-3, pentraxin-3, interleukin-6, and C-terminal telopeptide of type I. The best biomarkers in our evaluation (galectin-3 and pentraxin-3) were AAA diameter-independent. With the high AUC and AAA diameter correlation, the high-sensitive troponin I can be used as an independent prognostic biomarker of the upcoming heart complications in AAA patients. Authors recommend to add biomarkers as additional parameters to the current AAA patient management. Main addition value of biomarkers is in the assessment of the AAA with the smaller diameter. Elevated biomarkers can change the treatment decision, which would be done only based on AAA diameter size. The best way how to manage the AAA patients is to create a reliable predictive model of AAA growth and rupture risk. A created multiparameter model gives very promising results with the significantly higher efficiency compared with the use of the individual biomarkers.

Stability of total prostate-specific antigen and free prostate-specific antigen after 10 years' storage.

INTRODUCTION: PSA is a serine protease composed of 240 amino acids in a single polypeptide chain and is a routine parameter in prostate cancer diagnostics. The aim of our study was to test the long-term stability of tPSA and fPSA after 10 years' storage at -80°C. MATERIALS AND METHODS: We analyzed two aliquots from 55 serum samples. The first was assayed in routine testing at the time of establishing the diagnosis. The second was thawed for further testing after approximately 10 years' storage at -80°C. The mean of storage time was 10.41 years (min-max: 9.35-11.40 years). We compared the results of tPSA and fPSA. We calculated the fPSA/tPSA ratio and compared the results of clinical evaluation. Serum tPSA and fPSA levels were assayed using chemiluminescent kits Access Hybritech PSA and free PSA. All measurements were performed using the instrument UniCel® DxI 800. RESULTS: tPSA decreased 3.59% on average with a correlation r=0.9213, and fPSA increased at an average of 2.41% with a correlation r=0.9338. The fPSA/tPSA ratio increased 0.80% on average with a correlation r=0.9174. On clinical evaluation, five samples had fallen to a less malignant category and three samples had risen to a higher malignant category compared with the original results. CONCLUSION: The stability of tPSA and fPSA levels in serum is sufficient after 10 years' storage at -80°C. Calculation of the fPSA/tPSA ratio is not recommended due to the change in the category of malignancy of 15% of the samples.

PIVKA-II as a Potential New Biomarker for Hepatocellular Carcinoma - A Pilot Study.

AIM: The aim of this study was to evaluate the clinical contribution of protein induced by vitamin K absence (PIVKA-II) for hepatocellular carcinoma (HCC) diagnostics and compare it with alpha-foetoprotein (AFP), a routinely used tumour marker. MATERIALS AND METHODS: A total of 332 participants were enrolled in this study: 64 with HCC, 48 with liver metastases of colorectal cancer origin, 42 with liver cirrhosis and 178 healthy individuals. Serum levels of PIVKA-II were measured using the chemiluminescent assay of the Architect 1000i System (Abbott, USA) and AFP levels using the chemiluminescent assay by DxI 800 (Beckman Coulter, USA). RESULTS: PIVKA-II achieved better clinical sensitivity than AFP and the difference in this sensitivity was statistically significant. PIVKA-II achieved the best sensitivity (96.9%) in distinguishing between the HCC and control groups with the proposed cut-off value of 60 mAU/ml. CONCLUSION: Our recommendation is for addition of PIVKA-II to the routine panel of HCC tumour markers.

CEA, CA 15-3, and TPS as Prognostic Factors in the Follow-up Monitoring of Patients After Radical Surgery for Breast Cancer.

AIM: The aim of this study was to evaluate the ability of tissue polypeptide-specific antigen (TPS), carcinoembryonic antigen (CEA), and cancer antigen 15-3 (CA 15-3) to predict relapse in breast cancer patients, when the measurement of biomarkers is performed within 6 months after surgery. PATIENTS AND METHODS: Four hundred and seventy-two patients with breast cancer were evaluated. TPS, CEA, and CA 15-3 were measured in months 1, 3, and 6, after surgery. Disease recurrence was recorded between 7-12 months after surgery. Disease recurrence occurred in 60 patients, while 412 patients remained in recurrence-free status. RESULTS: TPS levels of the recurrence group differed statistically significantly in the first and sixth month after surgery compared to recurrence-free group (p=0.0339, AUC=0.6056; p<0.0001, AUC=0.7196). CEA and CA 15-3 measurements did not achieve a statistically significant difference for any month examined. CONCLUSION: TPS level in the sixth month after surgery is the best candidate biomarker to predict disease recurrence.

Circulating Growth and Angiogenic Factors and Lymph Node Status in Early-stage Breast Cancer - A Pilot Study.

AIM: To evaluate the possibility of selected biomarkers for breast cancer diagnostics and/or treatment monitoring, lymph node (LN) status determination and clinical decision regarding axillary node dissection. PATIENTS AND METHODS: Two hundred and eleven patients with malignant breast cancer and 42 age-matched healthy controls were enrolled. Serum insulin-like growth factor 1 (IGF1) and insulin-like growth factor binding protein 3 (IGFBP3) and plasma epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), osteoprotegerin (OPG) and osteopontin (OPN) were measured. We compared patients versus controls, patients with negative versus positive lymph node and patients with and without axillary lymph node dissection (ALND). RESULTS: We found elevated IGF1 and VEGF levels in patients with lymph node metastases compared to controls (p=0.0179 and p=0.0091, respectively) and in patients with ALND (p=0.0337 and p=0.0438, respectively). CONCLUSION: Circulating IGF1 and VEGF levels may predict the presence of lymph node metastases and help in the decision to avoid ALND in patients with early-stage breast cancer.

Predictive Value of Growth Factors and Interleukins for Future Liver Remnant Volume and Colorectal Liver Metastasis Volume Growth Following Portal Vein Embolization and Autologous Stem Cell Application.

BACKGROUND: Liver metastases occur in 60-80% of patients with colorectal carcinoma. The only potentially curative method is surgical resection, with an operability of 20-25%. The main reason for such low resectability is insufficient future liver remnant volume (FLRV). Portal vein embolization (PVE) alone is associated with failure in up to 40% of patients. A new method that could lead to acceleration of FRLV growth appears to be combination of PVE and application of hematopoietic stem cells (HSCs). The aim of our study was to evaluate the importance of growth factors and interleukins for FLRV growth after PVE and HSC application and also their possible effect on growth of colorectal liver metastases. PATIENTS AND METHODS: From June 2010 to July 2014, PVE was combined with application of adult HSCs in 16 primarily inoperable patients with colorectal liver metastases. We determined the serum levels of growth factors [hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), insulin-like growth factor 1 (IGF-1), insulin-like growth factor binging protein 3 (IGF-BP3), epidermal growth factor (EGF), transforming growth factor (TGFα), tumor necrosis factor (TNF)] and interleukins (IL2, -6, -8 and -10) at given time intervals by immunoanalytic methods. The growth of FLRV was evaluated by multidetector computed tomography at intervals of 1 week until sufficient growth of FLRV. RESULTS: We were able to perform radical surgery in 13 primarily inoperable patients (81.4%). The average FLRV growth was 23.1% (range=21.9-38.6%); from an initial FLRV of 30.5% (range=20.6-39%) to 40.1% (range=29-48%) before resection. The combination of levels of EGF, HGF, VEGF, IGF, TGFα and IL2,-6,-8 appears to be crucial for predicting operability. IL8 was statistically significant for the growth of colorectal liver metastases, and TGFα, IL2, and IL8 are important for a longer disease-free interval.

Searching for New Biomarkers and the Use of Multivariate Analysis in Gastric Cancer Diagnostics.

AIM: The first aim of this study was to search for new biomarkers to be used in gastric cancer diagnostics. The second aim was to verify the findings presented in literature on a sample of the local population and investigate the risk of gastric cancer in that population using a multivariant statistical analysis. PATIENTS AND METHODS: We assessed a group of 36 patients with gastric cancer and 69 healthy individuals. We determined carcinoembryonic antigen, cancer antigen 19-9, cancer antigen 72-4, matrix metalloproteinases (-1, -2, -7, -8 and -9), osteoprotegerin, osteopontin, prothrombin induced by vitamin K absence-II, pepsinogen I, pepsinogen II, gastrin and Helicobacter pylori for each sample. RESULTS: The multivariate stepwise logistic regression identified the following biomarkers as the best gastric cancer predictors: CEA, CA72-4, pepsinogen I, Helicobacter pylori presence and MMP7. CONCLUSION: CEA and CA72-4 remain the best markers for gastric cancer diagnostics. We suggest a mathematical model for the assessment of risk of gastric cancer.

Testing of a Novel Cancer Metastatic Multiplex Panel for the Detection of Bone-metastatic Disease - a Pilot Study.

BACKGROUND: Bone metastases develop in several malignancies (multiple myeloma, breast, prostate and lung carcinoma) and cause several complications. The aim of this study was to search for new biomarkers to use in monitoring of bone metastatic disease with the use of xMAP technology. PATIENTS AND METHODS: We assessed 62 oncological patients: 23 with no bone metastases, 28 with metastatic disease not having undergone therapy and 11 with metastatic disease treated by denosumab. Serum levels of dickkopf-related protein 1 (DKK1), growth differentiation factor-15 (GDF15), neuron-specific enolase (NSE), osteoprotegerin (OPG), osteonectin, periostin, tartrate-resistant acid phosphatase (TRAP5), tumor necrosis factor related weak inducer of apoptosis (TWEAK), chitinase-3-like protein 1 (YKL40), carboxy-terminal telopeptide (CTX) and procollagen type 1 N-terminal propeptide (PINP) were measured in each sample. RESULTS: The following biomarkers were observed to have significantly higher levels in the groups of patients with metastases in comparison to metastasis-free patients: GDF15 (p<0.0001), osteonectin (p=0.0311), TRAP5 (p<0.0046), TWEAK (p<0.0343) and YKL40 (p<0.0034). The changes in DKK1, NSE, OPG and periostin were not significant. CONCLUSION: We identified five new biomarkers: GDF15, osteonectin, TRAP5, TWEAK, and YKL40 as being promising markers for monitoring bone metastases.

A road toward better education in personalized medicine at universities and beyond.

Personalized medicine is likely to become a future direction of medicine. There is increased knowledge about gene functions in human health and disease and a rapid advance of biotechnologies. Personal genetic testing is available outside the medical room, as direct-to-consumer testing. There is concern about genetic literacy of general public and healthcare professionals which are to handle genetic results and their clinical interpretation. Education and training in personalized medicine and genetic/genomics/pharmacogenomics issues at different levels (high school, university, continuing medical education) is needed. Examples of innovated educational tools and curricula over the world are presented. The educational initiatives in the field of personalized medicine in the Czech Republic are followed from the very beginning.

Pre-graduate and post-graduate education in personalized medicine in the Czech Republic: statistics, analysis and recommendations.

The main goal of personalized medicine is the individualized approach to the patient's treatment. It could be achieved only by the integration of the complexity of novel findings in diverse "omics" disciplines, new methods of medical imaging, as well as implementation of reliable biomarkers into the medical care. The implementation of personalized medicine into clinical practice is dependent on the adaptation of pre-graduate and post-graduate medical education to these principles. The situation in the education of personalized medicine in the Czech Republic is analyzed together with novel educational tools that are currently established in our country. The EPMA representatives in the Czech Republic in cooperation with the working group of professionals at the Faculty of Medicine in Pilsen, Charles University in Prague have implemented the survey of personalized medicine awareness among students of Faculty of Medicine in Pilsen-the "Personalized Medicine Questionnaire". The results showed lacking knowledge of personalized medicine principles and students' will of education in this domain. Therefore, several educational activities addressed particularly to medical students and young physicians were realized at our facility with very positive evaluation. These educational activities (conferences, workshops, seminars, e-learning and special courses in personalized medicine (PM)) will be a part of pre-graduate and post-graduate medical education, will be extended to other medical faculties in our country. The "Summer School of Personalized Medicine in Plzen 2015" will be organized at the Faculty of Medicine and Faculty Hospital in Pilsen as the first event on this topic in the Czech Republic.