Fredrik Skjørten

Prognostic Factors in Patients With Metastatic (Stage D2) Prostate Cancer: Experience from the Scandinavian Prostatic Cancer Group Study-2

PURPOSE Nuclear texture reflects the overall structures of the chromatin organization. We recently reported the principles and prognostic importance of image analysis of nuclei from metastatic prostate cancer. Immunohistochemical up regulation of the adhesion molecule sialyl Lewis(x) is also reported to be a prognostic parameter. Presently we analyzed statistically the prognostic impact of these 2 new parameters compared to well-known clinical parameters in metastatic prostate cancer. MATERIALS AND METHODS Prognostic factors, such as sedimentation rate, alkaline and acid phosphatases, hemoglobin, testosterone, performance status, pain due to metastasis, T category, histological grade and patient age, were included in a multivariate Cox proportional hazards regression analysis based on 262 patients from the Scandinavian Prostatic Cancer Group Study-2. Extent of bone lesions, deoxyribonucleic acid ploidy, texture analysis and sialyl Lewis(x) molecules based on subsets of these 262 patients were also analyzed in the same multivariate model. RESULTS This test identified chromatin texture as the most important factor (p < 0.001), followed by reaction of the oligosaccharide sialyl Lewis(x) (p < 0.01). Among the routine clinical and laboratory data, sedimentation rate, alkaline phosphatase and hemoglobin (p < 0.05) showed prognostic importance. Performance status, pain due to metastasis and extent of bone lesions showed prognostic value in the univariate analysis (p < 0.05). CONCLUSIONS These data indicate that computerized nuclear texture analysis as well as up regulation of sialyl Lewis(x) molecules may be new important prognostic factors in metastatic prostate cancer. Furthermore the prognostic importance of sedimentation rate, alkaline phosphatase and hemoglobin was confirmed.

Prostatic intraepithelial neoplasia in surgical resections: Relationship to coexistent adenocarcinoma and atypical adenomatous hyperplasia of the prostate

High grade prostatic intraepithelial neoplasia (PIN) is associated with coincident prostate carcinoma, and has been considered to be a precursor of prostate carcinoma. Most studies on PIN have been performed on total prostatectomy or core needle biopsy specimens. Few reports deal with the occurrence of PIN in consecutive surgical resections, which is the objective of the current study.

Is prostatic intraepithelial neoplasia in the transition/central zone a true precursor of cancer? A long-term retrospective study in Norway.

Prostatic intraepithelial neoplasia (PIN) has been considered as a precursor of prostatic cancer. Few reports have dealt with the long-term follow-up of PIN lesions, and there is still a lack of proof that PIN is a true premalignant lesion. The objective of this study was to evaluate PIN in the transition/central zone as a marker for subsequent development of prostatic cancer. The PIN status of tissue specimens from 789 men without prostate cancer was determined in 508 transurethral resections and 281 transvesical prostatic enucleations. All slides were reviewed blind and independently by two pathologists. The patients were followed for an average of 11 years, and the incidence of subsequent cancer and cause-specific survival were analysed. Thirty-six cases of clinical prostatic cancer occurred among the cohort of 789 men through follow-up. No association between the presence of PIN in the transition/central zone and subsequent cancer development was found. There was also no difference in survival related to PIN status among the subsequent cancer patients.

Subpopulations of human lung alveolar macrophages: ultrastructural features.

Lung alveolar macrophages (LAM), obtained by bronchoalveolar lavage of healthy donors, were separated into four subfractions on discontinuous gradients of Percoll and subjected to light microscopic, transmission (TEM) and scanning electron microscopic (SEM) studies. Alveolar macrophage morphometric analysis was performed on cytocentrifuged preparations. TEM of subpopulations revealed considerable morphologic heterogeneity. By SEM, cells of the most dense (D) subfraction were small, round, and, typically, the surface was highly ruffled with small membrane pseudopods. Cells of the least dense subfraction (A) showed a low degree of membrane folding or filopodia and were often totally disorganized. In smokers, macrophages of fraction A had a greater area and perimeter compared with non-smokers, whereas the inverse relationship was observed for C and D cells. Also, the number of electron-dense inclusions and the level of acid phosphatase were higher in smokers than in non-smokers. Coupled with functional heterogeneity the morphologic differences described in this paper suggest that density-separated subpopulations of LAM may represent different stages of differentiation or maturation.

Nuclear texture analysis: A new prognostic tool in metastatic prostate cancer

This report describes the prognostic value of computerized nuclear texture analysis in metastatic prostate cancer. Seventy-seven patients with histologically verified prostate carcinomas and skeletal metastases were selected from a Scandinavian multicenter study (SPCG-2). Thirty-six therapy-resistant patients experienced objective progression and cancer-related death within 2 years after orchiectomy. Thirty patients responded well to orchiectomy, i.e., showed objective disease remission and no signs of progression during 3 years of follow-up. From this data set, 10 randomly chosen therapy-resistant and 10 randomly chosen therapy-sensitive carcinomas were used in our previous study to find the optimal combination of features that can discriminate between the two groups (Yogesan et al.: Cytometry 24:268-276, 1996). In addition to these two groups, 11 patients experienced stable disease or disease remission during the first year and a secondary progression during the second or third year of follow-up, with subsequent cancer-related death. Traditional clinical prognostic factors such as histopathological grading and serum markers could not discriminate between these groups of patients. Therefore, image analysis techniques based on texture analysis have been utilized in this study of prognosis of prostate cancer. Feulgen-stained monolayers of nuclei were prepared from paraffin-embedded material taken from the primary tumor before endocrine ablation. Four different textural features were selected from the training data set to calculate the discriminating function. This function separated the therapy-sensitive and the therapy-resistant patients with 87% accuracy in the independent data set. This study demonstrates that it is possible to predict tumor progression and survival for endocrine-ablated metastatic prostate carcinomas using computerized nuclear texture analysis on light microscopy images from prostate biopsies taken at the time of diagnosis.

Mechanism for antigen detection on deplasticized epoxy sections

The purpose of this investigation was to explain why deplasticizing of epoxy sections gives higher immunogold labeling than non-deplasticizing. The methods used were the following: (1) Comparison of the ratio of immunogold labeling of deplasticized and non-deplasticized sections with gold particles of different sizes and comparison of this ratio with respect to sections of different thickness, (2) the tilt method (Brorson et al., 1994). Human kidney tissue with amyloid A depositions, human fibrin, and human pituitary tissue were embedded, sections were deplasticized on grids, treated with anti-Aa, anti-fibrinogen or anti-ACTH (ACTH = adrenocorticotropic hormone), and reembedded on grids. Indications of significant antibody penetration were found only at the periphery of structures (ACTH-vesicles). This penetration was about 30 nm. The ratios of immunogold labeling of deplasticized and non-deplasticized sections were approximately 2, 5 and 1 for amyloid, fibrin and ACTH, respectively, and were independent of the gold particle size. No significant differences of gold labeling were found between thicker and thinner deplasticized epoxy sections regardless the gold particle size. No significant differences of gold labeling between deplasticized epoxy sections and LR-White sections were found on interior areas of ACTH-vesicles or amyloid A plaques. The increased labeling of deplasticized epoxy sections compared to normal epoxy sections seemed to be mainly a surface phenomenon. The practical significance of this observation is that deplasticizing of epoxy sections may be a better method for localizing antigens at the periphery of structures than the use of other resin embedding media. Deplasticizing of epoxy sections may be a method of choice in a pathological laboratory to detect antigens in routinely embedded tissues.

The theoretical relationship of immunogold labeling on acrylic sections and epoxy sections

The purpose of this study was to predict the ratio of immunogold labeling of LR-White sections and epoxy sections using theoretical methods. Tissues used in the experiments were pancreas, pituitary, kidney, thyroid and fibrin. Antigens used as test proteins were glucagon, somatostatin, thyroglobulin, chromogranin A, ACTH (adrenocorticotropt hormone), amyloid A and fibrinogen. These are proteins of different sizes. The quotient labelingLR-White/labelingepoxy was deduced theoretically and compared to calculations based on practical immunogold experiments. The theoretically deduced formula showed acceptable correlation to these calculations. This study gives a theory--expressed mathematically--for what is happening on the molecular level at the surface of resin sections in immunoelectron microscopy. The theory explains why acrylic resins normally are better suited for immunoelectron microscopy than epoxy sections, and indicates increased usefulness of epoxy sections when the diameter of the protein carrying the epitope decreases.

Immunoelectron microscopy on epoxy sections without deplasticizing to detect glomerular immunoglobulin and complement deposits in renal diseases

Twenty renal biopsies were studied by immunoelectron microscopy (IEM) after embedding in epoxy resin. Immunogold labeling for immunoglobulins and complement C3 was performed on the epoxy sections, which were not subjected to any kind of etching or deplasticizing prior to the immunolabeling. The concentration of accelerator, DMP‐30 (Tri (Dimethyl Amino Methyl) Phenol), was increased in the infiltration and embedding steps far beyond the values normally used to make immunolabeling of these antigens possible on epoxy sections. The sections were stained with tannic acid accompanied by uranyl acetate and lead citrate. Immunofluorescence (IF) for light microscopy was carried out on frozen sections of parallel tissue samples. Some cases with IgA‐nephritis demonstrated a higher sensitivity for IEM than IF, in the sense that smaller amounts of antigen were detectable with IEM. Ultrastructural preservation with this method was approximately the same as that usually seen on epoxy‐embedded material. By combining excellent immunolabeling with nearly optimal ultrastructural morphology in one procedure, this method is useful particularly in situations where the material available is limited, such as in studies of renal biopsies. As far as we know, this is the first time that immunoglobulins have been satisfactorily immunolabeled on epoxy sections without etching or deplasticizing.

The use of post‐embedding immunoelectron microscopy in the diagnosis of glomerular diseases

Fifty renal biopsies were studied by immunoelectron microscopy after embedding in a partly hydrophilic polyacrylic resin (LR White). Immunofluorescence studies were carried out on frozen sections of parallel tissue samples. Polyacrylic embedding gave good preservation of the renal ultrastructure and precise localization of immunoglobulin and C3c antibodies within glomerular electron‐dense deposits. Non‐specific staining of plasma proteins within vascular lumina could easily be detected. There was good correlation between immunoelectron and immunofluorescence microscopy. Immunoelectron microscopy is a very sensitive method, which can detect small amounts of antigen. More cases were, however, positive by immunofluorescence than by immunoelectron microscopy. This discrepancy may be explained by difference in sample size, and by difference in resolution of morphological details (electron microscopy versus fluorescence microscopy).

Histopathological grading and DNA ploidy as prognostic markers in metastatic prostatic cancer

The present study compares the prognostic potential of tumour grade and DNA ploidy status in patients with advanced-stage prostatic cancer. Two outcome groups were selected on the basis of time to progression and survival after orchiectomy. A poor-outcome group consisted of 32 therapy-resistant patients who experienced disease progression during the first year after orchiectomy and subsequently death due to prostatic cancer during the following year. A good-outcome group consisted of 27 therapy-responsive patients who showed disease regression and no signs of progression during a 3 year follow-up. The primary tumours were graded twice according to WHO and Gleason classification systems by two pathologists. Final agreement between the pathologists was obtained after a consensus meeting. The analysis revealed no prognostic importance of the two histological classification systems (P = 0.62 and P = 0.70) and disclosed weak inter- and intra-observer reproducibility (kappa < 0.70). DNA ploidy analyses were performed by image cytometry on formalin-fixed, paraffin-embedded samples of the primary tumours. Overall, 48% of the tumours were diploid, 20% tetraploid and 32% anueploid. DNA ploidy status did not discriminate between the two outcome groups (P = 0.46). Histological grade and DNA ploidy showed no prognostic importance in patients with prostatic cancer and skeletal metastases.