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Cytoglobin (Cygb) is a member of the hemoglobin family and is thought to protect against cellular hypoxia and oxidative stress. These functions may be particularly important in inflammation-induced cancer, e.g., in patients with ulcerative colitis (UC). In this study, we investigated the development of inflammation and tumors in a murine model of inflammation-induced colorectal cancer using a combined treatment of azoxymethane and dextran sulfate sodium. A bioinformatics analysis of genome-wide expression data revealed increased colonic inflammation at the molecular level accompanied by enhanced macroscopic tumor development in Cygb-deficient mice. Moreover, the expression of the UC-associated gene neurexophilin and PC-esterase domain family member 4 (Nxpe4) depended on the presence of Cygb in the inflamed colonic mucosa. Compared to wild type mice, RT-qPCR confirmed a 14-fold (p = 0.0003) decrease in Nxpe4 expression in the inflamed colonic mucosa from Cygb-deficient mice. An analysis of Cygb protein expression suggested that Cygb is expressed in fibroblast-like cells surrounding the colonic crypts. Histological examinations of early induced lesions suggested that the effect of Cygb is primarily at the level of tumor promotion. In conclusion, in this model, Cygb primarily seemed to inhibit the development of established microadenomas.

BACKGROUND: Fluoro-L-thymidine (FLT) is a positron emission tomography/computed tomography (PET/CT) tracer which reflects proliferative activity in a cancer lesion. The main objective of this prospective explorative study was to evaluate whether FLT-PET can be used for the early evaluation of treatment response in colorectal cancer patients (CRC) with liver metastases. Patients with metastatic CRC having at least one measurable (>1 cm) liver metastasis receiving first-line chemotherapy were included. A FLT-PET/CT scan was performed at baseline and after the first treatment. The maximum and mean standardised uptake values (SUVmax, SUVmean) were measured. After three cycles of chemotherapy, treatment response was assessed by CT scan based on RECIST 1.1.RESULTS: Thirty-nine consecutive patients were included of which 27 were evaluable. Dropout was mainly due to disease complications. Nineteen patients (70%) had a partial response, seven (26%) had stable disease and one (4%) had progressive disease. A total of 23 patients (85%) had a decrease in FLT uptake following the first treatment. The patient with progressive disease had the highest increase in FLT uptake in SUVmax. There was no correlation between the response according to RECIST and the early changes in FLT uptake measured as SUVmax(p = 0.24).CONCLUSIONS: No correlation was found between early changes in FLT uptake after the first cycle of treatment and the response evaluated from subsequent CT scans. It seems unlikely that FLT-PET can be used on its own for the early response evaluation of metastatic CRC.

Background: Dynamic contrast-enhanced computed tomography (DCE-CT) is a promising non-invasive method that provides the functional evaluation of the vascularity in normal and malignant tissue. The objectives of this consecutive study were to investigate the possible correlation between the perfusion characteristics of colorectal cancer liver metastases as examined by (DCE-CT) and the microvessel density of resected metastases. An additional aim was to investigate the correlation between the urokinase plasminogen activation receptor (uPAR) and perfusion values. Methods and findings: Eleven patients fulfilled the criteria for comparative analyses. The microvessel density values,uPAR level and the DCE-CT values were analysed. A perfusion index (PI) based on the measurement of arterial and portal flow (AF, PF) was defined as follows: PI=AF/ (AF+PF)%. The DCE-CT measurements were compared between metastatic and normal liver tissues. A Spearman correlation test was used for statistical analysis. The perfusion index and microvessel density values were significantly correlated (r=0.75; p=0.01). Furthermore, a higher volume of metastases significantly correlated with higher plasma levels of the uPAR forms (0.72 ≤ r ≥ 0.89; p < 0.05). Conclusions: DCE-CT may have the potential to measure the vascularity of colorectal liver metastases; however, the correlation between microvessel density and the perfusion values appears vague.

Aims: Breast cancer is one of the most common cancer diseases in women, with >1.67 million cases being diagnosed worldwide each year. In breast cancer, the sentinel lymph node (SLN) pinpoints the first lymph node(s) into which the tumour spreads, and it is usually located in the ipsilateral axilla. In patients with no clinical signs of metastatic disease in the axilla, an SLN biopsy (SLNB) is performed. Assessment of metastases in the SLNB, when using a conventional microscope, is performed by manually observing a metastasis and measuring its size and/or counting the number of tumour cells. This is done essentially to categorize the type of metastasis as macrometastasis, micrometastasis, or isolated tumour cells, which is used to determine which treatment the breast cancer patient will benefit most from. The aim of this study was to evaluate whether digital image analysis can be applied as a screening tool for SNLB assessment without compromising the diagnostic accuracy. Materials and results: Consecutive SLNBs from 135 patients with localized breast cancer receiving surgery in the period February to August 2015 were collected and included in this study. Of the 135 patients, 35 were received at the Department of Pathology, Rigshospitalet, Copenhagen University Hospital, 50 at the Department of Pathology, Zealand University Hospital, and 50 at the Department of Pathology, Odense University Hospital. Formalin-fixed paraffin-embedded tissue sections were analysed by immunohistochemistry with the BenchMark ULTRA Ventana platform. Rigshospitalet used a mixture of cytokeratin (CK) 7 and CK19, Zealand University Hospital used pancytokeratin AE1/AE3 and Odense used pancytokeratin CAM5.2 for detection of epithelial tumour cells. Slides were stained locally. SLNB sections were assessed in a conventional microscope according to national guidelines for SLNBs in breast cancer patients. The immunohistochemically stained sections were scanned with a Hamamatsu NanoZoomer-XR digital whole slide scanner, and the images were analysed with Visiopharm's software by use of a custom-made algorithm for SLNBs in breast cancer. The algorithm was optimized to the CK antibodies and the local laboratory conditions, on the basis of staining intensity and background staining. Conventional microscopy was used as the gold standard for assessment of positive tumour cells, and the results were compared with those from digital image analysis. The algorithm showed a sensitivity of 100% (that is, no false-negative slides were observed), including 67.2%, 19.2% and 56.1% of the slides from the three pathology departments being negative, respectively. This means that, on average, the workload could have been decreased by 58.2% by use of the digital SLNB algorithm as a screening tool. Conclusions: The SLNB algorithm showed a sensitivity of 100% regardless of the antibody used for immunohistochemistry and the staining protocol. No false-negative slides were observed, which proves that the SLNB algorithm is an ideal screening tool for selecting those slides that a pathologist does not need to see. The implementation of automated digital image analysis of SLNBs in breast cancer would decrease the workload in this context for examining pathologists by almost 60%.

Histologic scoring systems specific for primary sclerosing cholangitis (PSC) are not validated. We recently determined the applicability and prognostic value of three histological scoring systems in a single PSC cohort. The aim of this study was to validate their prognostic use and reproducibility across a multicenter PSC cohort. Liver biopsies from PSC patients were collected from seven European institutions. Histologic scoring was performed using the Nakanuma, Ishak, and Ludwig scoring systems. Biopsies were independently scored by six liver pathologists for interobserver agreement. The prognostic value of clinical, biochemical, and all three histologic scoring systems on predicting composite endpoints 1 (PSC-related death and liver transplantation), 2 (liver transplantation), and 3 (liver-related events), was assessed using univariable and multivariable Cox proportional hazards modeling. A total of 119 PSC patients were identified, and the median follow-up was 142 months. During follow-up, 31 patients died (20 PSC-related deaths), 31 patients underwent liver transplantation, and 35 patients experienced one or more liver-related events. All three staging systems were independent predictors of endpoints 2 and 3 (Nakanuma system: hazard ratio [HR], 3.16 [95% confidence interval (CI), 1.49-6.68] for endpoint 2 and HR, 2.05 [95% CI, 1.17-3.57] for endpoint 3; Ishak system: HR, 1.55 [95% CI, 1.10-2.18] for endpoint 2 and HR, 1.43 [95% CI, 1.10-1.85] for endpoint 3; Ludwig system: HR, 2.62 [95% CI, 1.19-5.80] for endpoint 2 and HR, 2.06 [95% CI, 1.09-3.89] for endpoint 3). Only the Nakanuma staging system was independently associated with endpoint 1: HR, 2.14 (95% CI, 1.22-3.77). Interobserver agreement was moderate for Nakanuma stage (κ = 0.56) and substantial for Nakanuma component fibrosis (κ = 0.67), Ishak stage (κ = 0.64), and Ludwig stage (κ = 0.62). Conclusion: We confirm the independent prognostic value and demonstrate for the first time the reproducibility of staging disease progression in PSC using the Nakanuma, Ishak, and Ludwig staging systems. The Nakanuma staging system—incorporating features of chronic biliary disease—again showed the strongest predictive value. (Hepatology 2017;65:907-919).

Familiarity with the structure and composition of normal tissue and an understanding of the changes that occur during disease is pivotal to the study of the human body. For decades, microscope slides have been central to teaching pathology in medical courses and related subjects at the University of Copenhagen. Students had to learn how to use a microscope and envisage three-dimensional processes that occur in the body from two-dimensional glass slides. Here, we describe how a PathXL virtual microscopy system for teaching pathology and histology at the Faculty has recently been implemented, from an administrative, an economic, and a teaching perspective. This fully automatic digital microscopy system has been received positively by both teachers and students, and a decision was made to convert all courses involving microscopy to the virtual microscopy format. As a result, conventional analog microscopy will be phased out from the fall of 2016.

BACKGROUND: The opportunity offered by whole slide scanners of automated histological analysis implies an ever increasing importance of digital pathology. To go beyond the importance of conventional pathology, however, digital pathology may need a basic histological starting point similar to that of hematoxylin and eosin staining in conventional pathology. This study presents an automated fluorescence-based microscopy approach providing highly detailed morphological data from unstained microsections. This data may provide a basic histological starting point from which further digital analysis including staining may benefit.METHODS: This study explores the inherent tissue fluorescence, also known as autofluorescence, as a mean to quantitate cardiac tissue components in histological microsections. Data acquisition using a commercially available whole slide scanner and an image-based quantitation algorithm are presented.RESULTS: It is shown that the autofluorescence intensity of unstained microsections at two different wavelengths is a suitable starting point for automated digital analysis of myocytes, fibrous tissue, lipofuscin, and the extracellular compartment. The output of the method is absolute quantitation along with accurate outlines of above-mentioned components. The digital quantitations are verified by comparison to point grid quantitations performed on the microsections after Van Gieson staining.CONCLUSION: The presented method is amply described as a prestain multicomponent quantitation and outlining tool for histological sections of cardiac tissue. The main perspective is the opportunity for combination with digital analysis of stained microsections, for which the method may provide an accurate digital framework.

The purpose of this study was to characterise growth patterns, proteolysis, and angiogenesis in colorectal liver metastases from chemonaive patients with multiple liver metastases. Twenty-four patients were included in the study, resected for a median of 2.6 metastases. The growth pattern distribution was 25.8% desmoplastic, 33.9% pushing, and 21% replacement. In 20 patients, identical growth patterns were detected in all metastases, but in 8 of these patients, a second growth pattern was also present in one or two of the metastases. In the remaining 4 patients, no general growth pattern was observed, although none of the liver metastases included more than two growth patterns. Overall, a mixed growth pattern was demonstrated in 19.3% of the liver metastases. Compared to metastases with pushing, those with desmoplastic growth pattern had a significantly up-regulated expression of urokinase-type plasminogen activator receptor (P=0.0008). Angiogenesis was most pronounced in metastases with a pushing growth pattern in comparison to those with desmoplastic (P=0.0007) and replacement growth pattern (P=0.021). Although a minor fraction of the patients harboured metastases with different growth patterns, we observed a tendency toward growth pattern uniformity in the liver metastases arising in the same patient. The result suggests that the growth pattern of liver metastases is not a random phenomenon.

Liver fibrosis involves excessive remodeling and deposition of fibrillar extracellular matrix (ECM) components, which leads to malfunction of the organ, causing significant morbidity and mortality. The aim of this study was to assess whether levels of a type V collagen fragment, the propeptide CO5-1230, indicate the amount of collagen deposited during liver fibrosis.A specific competitive enzyme-linked immunosorbent assay (ELISA) was developed to measure CO5-1230 levels. The sequence TAALGDIMGH located at the start of the C-terminal propeptide between amino acid position 1230' and 1239' (CO5-1230) of the α2 chain was selected as the immunogen. Monoclonal antibodies were raised against this fragment. An assay developed using the biotin-streptavidin system was evaluated in two rat models of liver fibrosis: bile duct ligation (BDL) and carbon tetrachloride (CCl(4))-treated rats, for up to 20 weeks.The ELISA was capable of measuring CO5-1230 in serum specifically, with an intra-assay variation of 3.46% and inter-assay variation of 5.09%. Mean CO5-1230 levels were significantly elevated in CCl(4) rats compared with controls [8 weeks: 57.4 ng/mL, controls 45.5 ng/mL (P = 0.0020); 12 weeks: 81.3 ng/mL, controls 50.2 ng/mL (P = 0.0020); 16 weeks: 85.1 ng/mL, controls 51 ng/mL (P = 0055); 20 weeks: 92 ng/mL, controls 47.8 ng/mL (P = 0.0033)]. CO5-1230 levels correlated with the total amount of collagen in sections from the injured livers, quantified from Sirius red stains (Spearman, R(2) = 0.5580). In BDL rats, serum levels of CO5-1230 were also elevated compared with controls [2 weeks: 160.1 ng/mL, controls 78.9 ng/mL (P = 0.0007); 4 weeks: 111.3 ng/mL, controls 62.2 ng/mL, (P = 0.0068)] and showed a linear correlation to the total collagen content (Spearman, R(2) = 0.3305).Increased serum levels of CO5-1230 were associated with the extent of collagen deposition in two different models of fibrotic processes in the liver. The data indicate that formation of type V collagen may be of value as a disease-specific diagnostic biomarker that reflects the total burden of disease. The amino acid sequence selected is located in the first 10 amino acids of the C-terminal propeptide section, which is a formation-specific region.

Background:Fibrosis is a central histological feature of chronic liver diseases and is characterized by the accumulation and reorganization of the extracellular matrix. The gold standard for assessment of fibrosis is histological evaluation of a percutaneous liver biopsy. Albeit a considerable effort have been invested in finding alternative non-invasive approaches, these have not been sufficiently succesfull to replace biopsy assessment.Aim:To identify the extracellular matrix proteins of interest, that as protein degradation fragments produced during extracellular matrix metabolism neo-epitopes, may be targeted for novel biochemical marker development in fibrosis. We used the recently proposed BIPED system (Burden of disease,Investigative,Prognostic,Efficacy andDiagnostic) to characterise present serological markers.Methods:Pubmed was search for keywords; Liver fibrosis, neo-epitopes, biomarkers, clinical trail, extra cellular matrix, protease, degradation, fragment.Results and Conclusion:Implementation of BIPED categorization in the development and validation of fibrosis biomarkers to simplify and standardize the use of existing and future biomarkers seems advantageous. In addition, a systematic use of the neo-epitope approach, i.e. the quantification of peptide epitopes generated from enzymatic cleavage of proteins during extracellular remodeling, may prove productive in the quest to find new markers of liver fibrosis.

AIM: To determine the efficacy of tacrolimus on clinical status, histopathological status and biochemical markers in patients with steroid refractory autoimmune hepatitis (AIH). METHODS: Retrospectively, clinical parameters, biochemistry and histology were obtained from patient records. RESULTS: Nine patients [8 females/1 male, median age 32 (range 16-64) years] were identified to have received tacrolimus for a median duration of 18 (12-37) mo. Before initiation of tacrolimus treatment the patients were maintained on a prednisolone dose of 20 mg daily (range 20-80 mg/d), which was tapered to 7.5 (5-12.5) mg/d (P = 0.004). Alanine aminotransferase and immunoglobulin-G concentrations decreased from 154 (100-475) to 47(22-61) U/L (P = 0.007), and from 16 (10-30.2) to 14.5 (8.4-20) g/L (P = 0.032), respectively. All patients showed improvement of the liver inflammatory activity, as determined by the Ishak score (P = 0.016), while the degree of fibrosis tended to decrease (P = 0.049). CONCLUSION: The use of low dose tacrolimus can lead to biochemical and histologic improvement of inflammation with no progression of the stage of fibrosis in patients with steroid refractory AIH. Low dose tacrolimus therapy also allows substantial reduction of prednisone dose.

CONCLUSIONS: Juvenile nasopharyngeal angiofibroma (JNA) is a rare tumor in young males, with a non-negligible potential for recurrence. Preoperative embolization is a safe procedure that diminishes the peroperative blood loss and the need for blood transfusion. The endoscopic approach was used with good results in JNA stage I and II (Chandler). OBJECTIVES: To estimate the incidence rate of JNA in the Danish population and to describe symptoms and treatment. PATIENTS AND METHODS: This was a national retrospective cohort study. All cases of JNA diagnosed in Denmark from 1981 to 2003 were identified. Data were extracted from medical records. RESULTS: Forty-five male (no female) JNA cases were identified. In 43 cases, clinical data were recovered. Median age was 15 years. The incidence rate in Denmark was 0.4 cases per million inhabitants per year and 3.7 cases per million males (aged 10-24) per year. All patients underwent surgery, and the endoscopic approach was increasingly being used. The embolization procedure proved to be safe and decreased the intraoperative blood loss statistically to 650 ml in the embolized group from an average of 1200 ml in the non-embolized group (p Udgivelsesdato: 2007-Mar

Background Fibrosis can be described as the excess deposition of extracellular matrix (ECM) components, such as collagens and proteoglycans. Fibrosis of the liver, which eventually leads to cirrhosis, is a major global health problem. Being able to measure fibrosis progression may enable timely preventative intervention. The aim of the current study was to investigate the utility of serum procollagen type I N-terminal propeptide (PINP) as a marker of hepatic fibrosis, as distinct from bone formation, during three different periods of fibrosis development following hepatic injury induced by bile duct ligation (BDL) in rats. Methods BDL was performed on 30 female Sprague-Dawley rats aged 6 months, and sham operations on 30 controls. Animals were killed after 14, 28, or 35 days. The extent of liver fibrosis was evaluated by quantitative histology after Sirus Red staining. Levels of serum PINP and osteocalcin (a marker solely for osteoblastic bone formation) were determined using ELISA at baseline and post termination. Results Collagen formation increased by 30% compared to 3% in sham-operated animals (P < 0.0001). PINP levels increased significantly in all BDL groups compared with baseline (14 days: baseline 13.9 ng/ml, termination 17.7 ng/ml, P = 0.047; 28 days: baseline 17.9 ng/ml, termination 26.2 ng/ml, P = 0.005; 35 days: baseline 18.0 ng/ml, termination 27.4 ng/ml P = 0.015, an increase of 52%). PINP levels did not change from baseline in the sham-operated rats, indicating that the increased PINP levels were due to hepatic injury. The bone-specific marker, osteocalcin, did not increase in either BDL or sham-operated rats. PINP measured in serum correlated to the extent of liver fibrosis as evaluated by quantitative histology (R2 = 0.42, P < 0.001). Conclusion PINP was associated with the development of liver fibrosis, but not bone formation, in mature rats subjected to BDL. Thus, PINP may be useful in studying the pathogenesis of liver fibrosis. However, caution should be applied when interpreting PINP levels in other disease states.

Background Microsatellite instability (MSI) refers to mutations in short motifs of tandemly repeated nucleotides resulting from replication errors and deficient mismatch repair (MMR). Colorectal cancer with MSI has characteristic biology and chemosensitivity, however the molecular basis remains unclarified. The association of MSI and MMR status with outcome and with thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) expression in colorectal cancer were evaluated. Methods MSI in five reference loci, MMR enzymes (hMSH2, hMSH6, hMLH1 and hPMS2), thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) expression were assessed in paraffin embedded tumor specimens, and associated with outcome in 340 consecutive patients completely resected for colorectal cancer stages II-IV and subsequently receiving adjuvant 5-fluorouracil therapy. Results MSI was found in 43 (13.8%) tumors. Absence of repair protein expression was assessed in 52 (17.0%) tumors, which had primarily lost hMLH1 in 39 (12.7%), hMSH2 in 5 (1.6%), and hMSH6 in 8 (2.6%) tumors. In multivariate analysis MSI (instable) compared to MSS (stable) tumors were significantly associated with lower risk of recurrence (hazard ratio (HR) = 0.3; 95% CI: 0.2–0.7; P = 0.0007) and death (HR = 0.4; 95% CI: 0.2–0.9; P = 0.02) independently of the TS and DPD expressions. A direct relationship between MSI and TS intensity (P = 0.001) was found, while there was no significant association with DPD intensity (P = 0.1). Conclusion The favourable outcome of MSI colorectal carcinomas is ascribed mainly to the tumor biology and to a lesser extent to antitumor response to 5-fluorouracil therapy. There is no evidence that differential TS or DPD expression may account for these outcome characteristics.

Background Collagen deposition and an altered matrix metalloproteinase (MMP) expression profile are hallmarks of fibrosis. Type IV collagen is the most abundant structural basement membrane component of tissue, which increases 14-fold during fibrogenesis in the liver. Proteolytic degradation of collagens by proteases produces small fragments, so-called neoepitopes, which are released systemically. Technologies investigating MMP-generated fragments of collagens may provide more useful information than traditional serological assays that crudely measure total protein. In the present study, we developed an ELISA for the quantification of a neoepitope generated by MMP degradation of type IV collagen and evaluated the association of this neoepitope with liver fibrosis in two animal models. Methods Type IV collagen was degraded in vitro by a variety of proteases. Mass spectrometric analysis revealed more than 200 different degradation fragments. A specific peptide sequence, 1438'GTPSVDHGFL'1447 (CO4-MMP), in the α1 chain of type IV collagen generated by MMP-9 was selected for ELISA development. ELISA was used to determine serum levels of the CO4-MMP neoepitope in two rat models of liver fibrosis: inhalation of carbon tetrachloride (CCl4) and bile duct ligation (BDL). The levels were correlated to histological findings using Sirius red staining. Results A technically robust assay was produced that is specific to the type IV degradation fragment, GTPSVDHGFL. CO4-MMP serum levels increased significantly in all BDL groups compared to baseline, with a maximum increase of 248% seen two weeks after BDL. There were no changes in CO4-MMP levels in sham-operated rats. In the CCl4 model, levels of CO4-MMP were significantly elevated at weeks 12, 16 and 20 compared to baseline levels, with a maximum increase of 88% after 20 weeks. CO4-MMP levels correlated to Sirius red staining results. Conclusion This ELISA is the first assay developed for assessment of proteolytic degraded type IV collagen, which, by enabling quantification of basement membrane degradation, could be relevant in investigating various fibrogenic pathologies. The CO4-MMP degradation fragment was highly associated with liver fibrosis in the two animal models studied.