Inflammation and uPAR-Expression in Colorectal Liver Metastases in Relation to Growth Pattern and Neo-adjuvant Therapy.

Proteolytic activity and inflammation in the tumour microenvironment affects cancer progression. In colorectal cancer (CRC) liver metastases it has been observed that three different immune profiles are present, as well as proteolytic activity, determined by the expression of urokinase-type plasminogen activator (uPAR).The main objectives of this study were to investigate uPAR expression and the density of macrophages (CD68) and T cells (CD3) as markers of inflammation in resected CRC liver metastases, where patients were neo-adjuvantly treated with chemotherapy with or without the angiogenesis inhibitor bevacizumab. Chemonaive patients served as a control group. The markers were correlated to growth patterns (GP) of liver metastases, i.e. desmoplastic, pushing and replacement GP. It was hypothesised that differences in proteolysis and inflammation could reflect tumour specific growth and therapy related changes in the tumour microenvironment. In chemonaive patients, a significantly higher level of uPAR was observed in desmoplastic liver metastases in comparison to pushing GP (p = 0.01) or replacement GP (p = 0.03). A significantly higher density of CD68 was observed in liver metastases with replacement GP in comparison to those with pushing GP (p = 0.01). In liver metastases from chemo treated patients, CD68 density was significantly higher in desmoplastic GP in comparison to pushing GP (p = 0.03). In chemo and bevacizumab treated patients only a significant lower CD3 expression was observed in liver metastases with a mixed GP than in those with desmoplastic (p = 0.01) or pushing GP (p = 0.05). Expression of uPAR and the density of macrophages at the tumour margin of liver metastasis differ between GP in the untreated patients. A higher density of T cells was observed in the bevacizumab treated patients, when desmoplastic and pushing metastases were compared to liver metastases with a mix of the GP respectively, however no specific correlations between the immune markers of macrophages and T cells or GP of liver metastases could be demonstrated.

Growth pattern of colorectal liver metastasis as a marker of recurrence risk.

Despite improved therapy of advanced colorectal cancer, the median overall survival (OS) is still low. A surgical removal has significantly improved survival, if lesions are entirely removed. The purpose of this retrospective explorative study was to evaluate the prognostic value of histological growth patterns (GP) in chemonaive and patients receiving neo-adjuvant therapy. Two-hundred-fifty-four patients who underwent liver resection of colorectal liver metastases between 2007 and 2011 were included in the study. Clinicopathological data and information on neo-adjuvant treatment were retrieved from patient and pathology records. Histological GP were evaluated and related to recurrence free and OS. Kaplan-Meier curves, log-rank test and Cox regression analysis were used. The 5-year OS was 41.8% (95% CI 33.8-49.8%). Growth pattern evaluation of the largest liver metastasis was possible in 224 cases, with the following distribution: desmoplastic 63 patients (28.1%); pushing 77 patients (34.4%); replacement 28 patients (12.5%); mixed 56 patients (25.0%). The Kaplan-Meier analyses demonstrated that patients resected for liver metastases with desmoplastic growth pattern had a longer recurrence free survival (RFS) than patients resected for non-desmoplastic liver metastases (p=0.05). When patients were stratified according to neo-adjuvant treatment in the multivariate Cox regression model, hazard ratios for RFS compared to desmoplastic were: pushing (HR=1.37, 95% CI 0.93-2.02, p=0.116), replacement (HR=2.16, 95% CI 1.29-3.62, p=0.003) and mixed (HR=1.70, 95% CI 1.12-2.59, p=0.013). This was true for chemonaive patients as well as for patients who received neo-adjuvant treatment.

[Konrad Birkhaug and the BCG vaccine]. / Konrad Birkhaug og BCG-vaksinen.

Konrad Elias Birkhaug (1892-1980) was born in Bergen to Elisa Marie Skorge and Karl Anders Birkhaug, a policeman, as the sixth of their ten children. After a period as a laboratory assistant at Bergen Municipal Hospital, he emigrated to USA in 1911. He graduated from the medical school of Johns Hopkins University in 1924. After residency in bacteriology, he became professor at Rochester University in 1927 and head of its laboratory of bacteriology. From 1932 to 1935 he worked as a senior scientist at the Institute Pasteur in Paris and from 1935 to 1945 as a fellow of the Chr. Michelsen Institute in Bergen. From 1937 he also headed the National Laboratory for production of BCG vaccine. After active involvement in the resistance movement against the Nazis during the Second World War, he returned to the USA in 1946 as head of the BCG laboratory of the State of New York in Albany. He retired in 1953 and settled in Bergen, where he died in 1980. Birkhaug is one of the pioneers in the research on immunity reactions to tuberculosis infection and BCG vaccination. He is also known for producing the first antiserum against erysipelas, which was used from 1927 until sulphonamides were discovered. In addition to his international scientific publications, he wrote two books in Norwegian, his autobiography and a book about the German eradication of a small fishermen's village in Western Norway during the Second World War.

[Magnus Haaland--a pioneer in international cancer research]. / Magnus Haaland--en pioner i internasjonal kreftforskning.

Pathologist and bacteriologist Magnus Haaland (1876-1935) was a pioneer in European experimental cancer research. For eight years he worked in some of the most prominent research institutions in France, Germany and England. He worked with Elie Metschnikow and Amedée Borrel at Institut Pasteur in Paris, with Paul Ehrlich in Frankfurt am Main and with Erwin Bashford at the Imperial Cancer Research Fund in London. From 1911 to his death in 1935, he was the head of the Gade Institute in Bergen. He became a pioneer in Norwegian bacteriology research and made particular contributions to the eradication of typhoid fever in Norway. His main scientific work was on the progression of experimental transplantable tumours in mice, patterns of metastasis, and experimental hyperthermia treatment. He gave the first description of neoplastic reticuloses in mice.

[Anatomist, pathologist and philanthropist]. / Anatom, patolog og donator.

Fredrik Georg Gade (1855-1933) was born in Bergen as the eldest son of a merchant and politician. He graduated from the University of Oslo in 1880. After clinical residency and training in anatomy and pathology at the National Hospital in Oslo, he worked in several of the most outstanding medical research institutions in Continental Europe, including the institutes of Robert Koch and Carl Friedländer in Berlin, Carl Weigert in Frankfurt, the pathologists and anatomists Victor Cornil, Louis-Antoine Ranvier and Louis Charles Malassez in Paris. Gade was associate professor (prosector) of anatomy in Oslo from 1897 to 1906 and also the editor of the Norwegian Medical Journal (Norsk Magazin for Laegevidenskaben). He was also one of the pioneers of cancer statistics in Norway. In addition to his scientific publications, he wrote extensively on political and cultural issues. Struck by serious illness he donated most of his family fortune to establish an institute for pathology in his home town Bergen, which opened in 1912 under the name Dr. med. F.G. Gades Pathologiske institutt. It later became one of the pillars of the Medical Faculty when the University of Bergen was established in 1946 (now: The Gade Institute, University of Bergen).

Invasiveness in vitro and biological markers in human primary glioblastomas.

Invasion of spheroids from 20 human primary glioblastomas into precultured fetal rat brain tissue in culture has been studied and quantified. Between 30 and 98 percent of the normal brain tissue was destroyed by invading glioma cells within 4 days. The degree of invasion did not correlate with patient survival. A slightly higher invasiveness and shorter survival was seen in tumors with EGF receptor overexpression, and the opposite pattern was found for tumors with a TP53 mutation. The degree of invasiveness in vitro was far higher than would be expected from the dynamics of clinically observed tumor spread. This suggests that mechanisms suppressing invasion may be operative in the normal brain; alternatively the differences may be due to a higher permissiveness of the fetal brain tissue for invasion in vitro.

Comparison of a linear miniature ultrasound probe and a radial-scanning echoendoscope in TN staging of esophageal cancer.

BACKGROUND: Endoscopic ultrasonography is a precise method for TN staging of esophageal cancer. We explored the staging properties of a linear miniprobe as compared with a radial-scanning echoendoscope. METHODS: Sixty-eight patients with esophageal cancer underwent preoperative TN staging using a 20-MHz linear miniprobe and a 7.5/12-MHz radial-scanning echoendoscope. Tumor stage was verified by surgery and/or histology. RESULTS: T and N stages were verified in 53 and 54 patients, respectively. T-staging accuracy using the echoendoscope was 70%. The high-frequency miniprobe could not differentiate between T3 and T4 tumors, but both systems had an accuracy of 87% in discriminating between T1, T2, and T3/4 stages. With traversable tumors, the accuracy of N staging was significantly better with the echoendoscope than with the miniprobe (90% vs. 48%, P = 0.008). CONCLUSIONS: The two endosonographic systems had similar accuracy for assessing transmural tumor growth, but the echoendoscope was superior in staging advanced transmural tumors and in predicting lymph node metastasis with traversable tumors.

Invasiveness by lacZ transfected non-small-cell lung cancer cells into human bronchial tissues in vitro.

To facilitate the detection of invading tumor cells in a three dimensional coculture assay in vitro, the reporter gene Escherichia coli beta-galactosidase (lacZ), was transfected into a human large-cell lung carcinoma cell line GaL23. Multicellular spheroids initiated from the transfected cell line, GaL23LZ, were confronted with fragments of human bronchial tissue differing in their surface composition. While an intact surface epithelium was found to obstruct both adhesion and invasion of tumor cells, an exposed basal lamina augmented adhesion, migration and invasion of tumor cells into the normal tissue. Tumor cells, migrating on the surface of the bronchial fragments, were found to migrate between the epithelial cells and the basal lamina. Fibroblast covered stromal fragments, derived from resected non-small cell lung cancers, were found to be more edible to the invading tumor cells than subepithelial stromal fragments from normal bronchi. The lacZ transfection made it possible to quantitatively analyze the invasive process. While the transfection neither changed the invasive ability of the tumor cells in vitro or in vivo nor their growth pattern in monolayers, three dimensional growth represented by spheroid morphology and clonogenicity in soft agar was significantly changed. This model offers an in vitro system to study qualitative and quantitative aspects of tumor-host relationships in a complex microenvironment which has several similarities to the in vivo situation.

Dynamic determination of human glioma invasion in vitro.

OBJECT: The goal of this study was to evaluate whether there is any relationship between survival of patients with brain tumor and tumor proliferation or tumor invasion in vitro. METHODS: Samples of freshly resected brain tumors from 14 patients with glioblastoma multiforme (GBM) were directly grown as three-dimensional multicellular spheroids. The tumor spheroids were cocultured with fetal rat brain cell aggregates (BCAs), used to represent an organotypical normal brain tissue model. Before the coculture, the tumor spheroids and the BCAs were stained with two different carbocyanine dyes, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) and 3,3'-dioctadecycloxacarbocyanine perchlorate (DiO), respectively. During the coculture, confocal laser scanning microscopy allowed a sequential analysis of tumor cell invasion by visualizing dynamic aspects of the invasive process. Single cocultures were examined at three different time points (24, 48, and 96 hours). During the observation period there was a change in the structural morphology of the cocultures, with a progressive decrease in BCA volume. Furthermore, the scanning confocal micrographs revealed a bidirectional movement of tumor cells and normal cells into brain and tumor tissue, respectively. It is also shown that there is a considerable variation in the rate of BCA destruction in cocultures of glioma spheroids generated directly from biopsy specimens. This variation is seen both between spheroids generated from the same biopsy as well as between spheroids that are grown from different biopsy specimens. Cell proliferation measured by Ki-67 immunohistochemical analysis of biopsy samples obtained in the same patients revealed a correlation between tumor cell proliferation and tissue destruction of the BCAs, as determined by a reduction in BCA volume (p = 0.0338). No correlation was found when survival was related to the same parameters (p > 0.05). CONCLUSIONS: The present work provides a model for quick and efficient assessment of dynamic interactions between tumor and normal brain tissue shortly after surgery.

Non-small-cell lung carcinoma cells invade human bronchial mucosa in vitro.

To study invasion of lung cancer in vitro a novel three-dimensional coculture assay consisting of living human tissues has been developed. Multicellular spheroids initiated from a new large-cell lung carcinoma cell line (GaL23), found to be invasive in immunodeficient mice, were confronted with precultured bronchial fragments derived from mucosal biopsies obtained during routine fiberoptic bronchoscopy. The bronchial fragments consist of a stromal core with scattered fibroblasts covered by a continuous surface epithelium resting on a basal lamina. During the first 2 wk of confrontation, a gradual retraction of the bronchial epithelium with subsequent adhesion of the tumor cells to the underlying basal lamina occurred. The following week, a limited invasion of tumor cells into the bronchial stroma was seen. To facilitate the entrance of tumor cells through the mucosal surface, the surface epithelium was removed prior to coculture by ethylenediaminetetraacetic acid (EDTA) buffer treatment. Upon confrontation, GaL23 cells then rapidly attached to and migrated on the exposed basal lamina and an increasing number of tumor cells was seen in the stroma during the first week of culture. This model offers opportunities for studying mechanisms of lung cancer adhesion, migration, and invasion using human bronchial mucosa as the natural target tissue.

Expression of annexin II in glioma cell lines and in brain tumor biopsies.

Annexin II is a calcium and phospholipid binding protein and a substrate for protein-tyrosine kinases. Increased levels of annexin II are observed in various cancer cells and tissues, and the molecule has been proposed as a marker of malignancy in vivo. Annexin II was expressed in four glioma cell lines (D-54MG, D-37MG, U251MG and GaMG), as determined by Western blot analyses, immunofluorescence staining and flow cytometric measurements. In addition, annexin II expression was also found in cryostat sections obtained from 15 consecutive brain tumor biopsies: Ten were histologically classified as glioblastomas, one as an astrocytoma, two as meningiomas and two as brain metastases. Cultured spheroids from the glioma cell lines and from three of the glioblastoma biopsies showed lower levels of annexin II, than found in the monolayers of the cell lines and in the freshly cut biopsies. The annexin II expression of the cell lines were not found to be related to their proliferative, migratory or invasive properties. These findings indicate that although annexin II may serve as a marker of malignancy in vivo, its expression can be reduced in vitro, and appear unrelated to malignant features of glioma cell lines.

Variation in cell yield and proliferative activity of positive selected human CD34+ bone marrow cells along the circadian time scale.

Variations in cell yield and proliferative activity of human bone marrow (BM) progenitor cells were determined with flow cytometry along the 24-h (circadian) time scale. Equal volumes of BM were aspirated every 5 h, altogether 5 times in 5 healthy men. An average 6-fold higher yield of positive selected CD34+ cells occurred in each subject when BM was aspirated during the daytime and late afternoon, while a lower yield occurred during the night. Using all CD34+ cell yield data normalized to percentage of mean, a significant time-effect was found by ANOVA (p = 0.02) and a significant circadian rhythm was detected by the least-squares fit of a 24 h cosine (p = 0.02). The 95% confidence limits of the acrophase (time of highest values) were computed to be at midday between 10:24 and 14:48 h. A highly significant correlation (p = 0.001) was found between proliferation of positive selected CD34+ cells and the more mature myeloid precursor cells from the same BM aspirates, suggesting a common temporal pattern along the circadian time scale. However, no correlation was demonstrated between proliferation and cell yield of CD34+ selected cells, suggesting that mechanisms other than variation in proliferation may cause the circadian rhythm in stem cell yield. These circadian variations in stem cell yield and proliferation suggest that proper timing within 24 h may potentially be important regarding outcome from progenitor cell harvesting and treatment with haematopoietic growth factors.

Circadian cell cycle variations of erythro- and myelopoiesis in humans.

By use of a multiparameter flow cytometric method with specific surface markers, circadian (24-h) variations in cell cycle distribution have been studied in 19 healthy male volunteers by sampling bone marrow (BM) every 4-5 h during 24-h periods. Admixture of peripheral blood during the sampling was specifically adjusted for, and the fractions of cells in DNA synthetic phase were measured for different hemopoietic cell lineages. Significant circadian variations in DNA S-phase were demonstrated both in myelo- and erythropoiesis of the human BM, with 75% (myeloid) and 80% (erythroid) of the volunteers showing highest activity (values) of DNA S-phase during the day and lowest activity (values) between midnight and 04:00 h. A temporal relationship in the circadian variation of S-phase and G2/M-phase was demonstrated between the myeloid and erythroid cell lineages. The highest fractions of S-phase cells were found in erythropoiesis, while the highest circadian stage dependent variation was found in myelopoiesis. The existence of a similar phasing in DNA synthetic activity for myelopoietic and erythropoietic cells in the human bone marrow indicates that the circadian rhythmicity of hemopoiesis may be caused by a common regulatory mechanism. These findings may be relevant with regard to optimizing the use of cytotoxic drugs and hemopoietic growth factors by taking into consideration the intrinsic (endogenous) circadian variation in proliferative activity of human BM subpopulations.

Local spread of malignant neuroepithelial tumors.

A short review of invasiveness of primary malignant neoplasms in the nervous system is given. Invasiveness implies progressive spread and destruction locally, which eventually leads to a fatal outcome in the patient. In particular, the malignant cells are able to rapidly migrate over large parts of the brain. This process includes the capacity to adhere to a substratum, usually constituted by the various components of the extracellular matrix, followed by detachment and migration. Anatomical structures and local regulatory factors in the brain influence the direction and extent of this migration. Several model systems are now available for monitoring the aggressiveness of such tumours both in vivo and in vitro, and different phenotypic properties characteristic of invasive cells have been elucidated. Although still in its infancy, and currently as an experimental approach, anti-invasive therapy may in the future be an interesting alternative to conventional chemotherapy of brain tumours.

[Medical and natrual scientists behind the museum which became the University of Bergen]. / Leger og naturvitere bak museet som ble til Universitetet i Bergen.

The University of Bergen celebrated its 50th anniversary in 1996. The University was built on an earlier institution, the Bergen Museum, founded in 1825. From 1880 until the beginning of this century the Museum gradually developed into an institution for research and higher education. The article describes work done by some of the key persons involved in this process, mainly medical and natural scientists. In particular, their international relations and ability to cooperative with other scientists were crucial factors in establishing the pillars of the later University of Bergen. Today, the University of Bergen has nearly 18,000 students and seven faculties, and is a highly internationalised institution.

Nonadhesive stationary organ culture of human bronchial mucosa.

The supply of fresh bronchial tissue from human donors for in vitro culture is limited. Routine fiberoptic bronchoscopy offers a safe and easy procedure for obtaining minor biopsies and we wanted to see if the material provided could be used for organ culture by using a simple liquid overlay technique. Bronchial biopsies were cut into fragments 400-500 microns and kept immersed in a standard serum-supplemented medium for 40 days. An agar base prevented adhesion of the tissue. By light and electron microscopy it was shown that the tissue fragments had a differentiated epithelium at their surface throughout the culture period. An outgrowth of epithelial cells on the scaffold of the exposed stroma, covering the surface of the whole fragment, occurred within the first 5 days of culture. This epithelium was partly ciliated, 2-4 cell layers thick with squamous and cuboidal cells and expressed epithelial markers (cytokeratin and Ber-Ep4). The amount of cilia increased during the first 15 days of culture. The epithelium rested on a neosynthesized basement membrane as visualized by electron microscopy and immunohistochemistry with antibodies directed against collagen IV, laminin, and fibronectin. The central stroma consisted of loose connective tissue with fibroblasts. This simple tissue culture model combines maintenance and neoformation of bronchial epithelium on top of a living natural substrate, thus enabling direct biological studies on clinical biopsy material under perfectly viable conditions.

Flow cytometric measurement of DNA S-phase in human bone marrow cells: correcting for peripheral blood contamination.

The relationship between bone marrow (BM) cells with S-phase DNA content and the amount of peripheral blood contamination estimated as percentage lymphocytes+monocytes (L+MO) present in BM samples has been investigated in a total of 136 BM aspirates and biopsy expellates from 35 hematologically healthy individuals. A significant negative correlation was demonstrated between total, erythroid and myeloid BM cells in S-phase and the percentage of L+MO in the aspirates (r=0.84, 0.57 and 0.49, respectively; p<0.0001). Based on the equation of the slope of the regression line, a correction formula adjusting the measured value of BM cells in S-phase to varying amounts of L+MO percentage has been worked out for the total and erythroid BM cells. In contrast, highly proliferating myelomonocytic cells and CD34+ cells did not show any significant correlation between cells in S-phase and percentage L+MO, indicating that peripheral blood contamination of BM aspirates estimates the degree of peripheral blood contamination, as well as make possible a correct estimation of the DNA synthesis of several BM populations. The method is especially applicable when frequent BM sampling is required.

Circadian cell kinetics in humans. Aspects related to cancer chemotherapy.

A prerequisite for the investigation of cell kinetics, and especially circadian cell kinetics, has been the development from the 1950s and onwards of several methods for studying kinetic parameters in different mammalian tissues. A large number of such studies have subsequently taken place in the rodent, mostly as non-circadian experiments, but also a large number of studies have now documented on circadian proliferative rhythms in many different murine tissues. Results from cytokinetic studies in the human have also accumulated through the years. Of special interest has been the demonstration of temporal variations in rapidly proliferating tissues studies as the bone marrow and gut mucosa relative to cytotoxic anticancer therapy. Analyses of proliferation in human tumours have also indicated rhythms in malignant solid tumours. Thus, these studies have demonstrated that there exist rhythms in bone marrow and gut cytokinetics which increases the likelihood that certain times of day will be less toxic for the administration of cytotoxic drugs. Furthermore, optimizing anticancer therapy according to a circadian schedule may also increase the tumour cell death rat, due both to an indirect dose-escalating effect and a direct increased tumour effect.