The novel collagen matrikine, endotrophin, is associated with mortality and cardiovascular events in patients with atherosclerosis.

BACKGROUND: Rupture of atherosclerotic plaques is the major cause of acute cardiovascular events. The biomarker PRO-C6 measuring Endotrophin, a matrikine of collagen type VI, may provide valuable information detecting subjects in need of intensified strategies for secondary prevention. OBJECTIVE: In this study, we evaluate endotrophin in human atherosclerotic plaques and circulating levels of PRO-C6 in patients with atherosclerosis, to determine the predictive potential of the biomarker. METHODS: Sections from the stenotic human carotid plaques were stained with the PRO-C6 antibody. PRO-C6 was measured in serum of patients enrolled in the Carotid Plaque Imagining Project (CPIP) (discovery cohort, n = 577) and the innovative medicines initiative surrogate markers for micro- and macrovascular hard end-points for innovative diabetes tools (IMI-SUMMIT, validation cohort, n = 1,378). Median follow-up was 43 months. Kaplan-Meier curves and log-rank tests were performed in the discovery cohort. Cox proportional hazard regression analysis (HR with 95% CI) was used in the discovery cohort and binary logistic regression (OR with 95% CI) in the validation cohort. RESULTS: PRO-C6 was localized in the core and shoulder of the atherosclerotic plaque. In the discovery cohort, PRO-C6 independently predicted future cardiovascular events (HR 1.089 [95% CI 1.019 -1.164], p = 0.01), cardiovascular death (HR 1.118 [95% CI 1.008 -1.241], p = 0.04) and all-cause death (HR 1.087 [95% CI 1.008 -1.172], p = 0.03). In the validation cohort, PRO-C6 predicted future cardiovascular events (OR 1.063 [95% CI 1.011 -1.117], p = 0.017). CONCLUSION: PRO-C6 is present in the atherosclerotic plaque and associated with future cardiovascular events, cardiovascular death and all-cause mortality in two large prospective cohorts.

Activation of immune responses against the basement membrane component collagen type IV does not affect the development of atherosclerosis in ApoE-deficient mice.

Oxidation of low-density lipoprotein (LDL) in the arterial extracellular matrix results in malondialdehyde (MDA)-modifications of surrounding matrix proteins. We have recently demonstrated an association between high levels of autoantibodies against MDA-modified collagen type IV and risk for development of myocardial infarction. Collagen type IV is an important component of the endothelial basement membrane and influences smooth muscle cell function. We hypothesized that immune responses against collagen type IV could contribute to vascular injury affecting the development of atherosclerosis. To investigate this possibility, we induced an antibody-response against collagen type IV in apolipoprotein E (Apo E)-deficient mice. Female ApoE-/- mice on C57BL/6 background were immunized with α1α2 type IV collagen chain peptides linked to the immune-enhancer PADRE, PADRE alone or PBS at 12 weeks of age with three subsequent booster injections before the mice were killed at 23 weeks of age. Immunization of PADRE alone induced autoantibodies against PADRE, increased IL-4 secretion from splenocytes and reduced SMC content in the subvalvular plaques. Immunization with peptides of α1α2 type IV collagen chains induced a strong IgG1antibody response against collagen type IV peptides without affecting the distribution of T cell populations, plasma cytokine or lipid levels. There were no differences in atherosclerotic plaque development between collagen α1α2(IV)-PADRE immunized mice and control mice. Our findings demonstrate that the presence of antibodies against the basement membrane component collagen type IV does not affect atherosclerosis development in ApoE-/- mice. This suggests that the association between autoantibodies against collagen type IV and risk for myocardial infarction found in humans does not reflect a pathogenic role of these autoantibodies.

A biomarker of collagen type I degradation is associated with cardiovascular events and mortality in patients with atherosclerosis.

OBJECTIVE: Atherosclerosis is characterized by accumulation of lipids, cells and extracellular matrix (ECM) proteins in the arterial wall. Collagen type I (COL1), a component of the arterial ECM, is cleaved by matrix metalloproteinases (MMPs) and known to be remodelled in atherosclerosis. We explored whether the MMP-mediated COL1 biomarker, C1M, was associated with cardiovascular events, cardiovascular mortality and all-cause mortality in a large prospective cohort of patients with known atherosclerosis. METHODS: Serum from 787 patients who underwent a carotid endarterectomy was included. Circulating levels of C1M were measured in serum. A total of 473 patients were followed for 6 years after surgery. Associations between C1M and incidence of cardiovascular events, cardiovascular mortality and all-cause mortality were assessed by Kaplan-Meier curves and Cox regression analysis. RESULTS: A total of 101 (21.4%) patients suffered from nonfatal cardiovascular events during the follow-up period, and 64 (13.5%) patients died. Of these, 39 (60.9%) died from cardiovascular diseases. Patients with C1M levels above the median were significantly associated with cardiovascular events, cardiovascular mortality and all-cause mortality (P < 0.001, P = 0.004 and P < 0.001, respectively). C1M was included in the final model for prediction of cardiovascular events (HR 2.15, 95% CI 1.40-3.32, P = 0.001), cardiovascular mortality (HR 2.20, 95% CI 1.07-4.51, P = 0.031) and all-cause mortality (HR 2.98 95% CI 1.67-5.33, P = < 0.001). CONCLUSIONS: In patients with atherosclerotic carotid lesions, high levels of C1M predicted cardiovascular events, cardiovascular mortality and all-cause mortality. These findings emphasize the importance of remodelling mechanisms in atherosclerosis that are now becoming more and more explored.

Autoantibodies against aldehyde-modified collagen type IV are associated with risk of development of myocardial infarction.

BACKGROUND: Oxidation of LDL particles entrapped in the extracellular matrix of the arterial wall is a key factor in the development of atherosclerosis. Lipid oxidation products, such as malondialdehyde (MDA), react with surrounding extracellular matrix proteins and cause modifications that are recognized by the immune system. MDA modification of collagen type IV is increased in carotid lesions from symptomatic patients and correlates with autoantibodies against MDA-modified collagen type IV in plasma. OBJECTIVE: The aim of this study was to determine whether autoantibodies against MDA-modified collagen type IV predict risk of development of myocardial infarction (MI). METHODS: Plasma levels of MDA-modified collagen type IV IgM and IgG antibodies were analysed by enzyme-linked immunosorbent assay in 385 subjects with incident MI during 13 years of follow-up and 410 age- and sex-matched controls in the Malmö Diet and Cancer study. RESULTS: MDA-modified collagen type IV IgG levels were higher in cases with incident MI than in controls. Subjects in the highest tertile of MDA-modified collagen type IV IgG had an increased risk of MI (hazard ratio 1.56, 95% confidence interval 1.22-2.00, P for trend 0.0004). This association remained significant after adjusting for factors included in the Framingham risk score and diabetes. High levels of MDA-collagen type IV IgG were associated with increased carotid intima-media thickness and elevated plasma levels of matrix metalloproteinase 10 and 12. CONCLUSIONS: Immune responses against MDA-modified collagen type IV are associated with more severe carotid disease and increased risk of MI. These immune responses may reflect LDL oxidation in the artery wall, but could also affect the atherosclerotic disease process.

Human Lipoproteins at Model Cell Membranes: Effect of Lipoprotein Class on Lipid Exchange.

High and low density lipoproteins (HDL and LDL) are thought to play vital roles in the onset and development of atherosclerosis; the biggest killer in the western world. Key issues of initial lipoprotein (LP) interactions at cellular membranes need to be addressed including LP deposition and lipid exchange. Here we present a protocol for monitoring the in situ kinetics of lipoprotein deposition and lipid exchange/removal at model cellular membranes using the non-invasive, surface sensitive methods of neutron reflection and quartz crystal microbalance with dissipation. For neutron reflection, lipid exchange and lipid removal can be distinguished thanks to the combined use of hydrogenated and tail-deuterated lipids. Both HDL and LDL remove lipids from the bilayer and deposit hydrogenated material into the lipid bilayer, however, the extent of removal and exchange depends on LP type. These results support the notion of HDL acting as the 'good' cholesterol, removing lipid material from lipid-loaded cells, whereas LDL acts as the 'bad' cholesterol, depositing lipid material into the vascular wall.

Plasma stem cell factor levels are associated with risk of cardiovascular disease and death.

OBJECTIVE: Stem cell factor (SCF) is a key growth factor for several types of stem and progenitor cells. There is experimental evidence that such cells are of importance for maintaining the integrity of the cardiovascular system. We investigated the association between circulating levels of SCF and risk for development of cardiovascular events and death. METHODS: SCF was analysed by the proximity extension assay technique in plasma from 4742 subjects participating in the Malmö Diet and Cancer Study. Cardiovascular events and death were monitored through national registers with a mean follow-up time of 19.2 years. RESULTS: Subjects with high baseline levels of SCF had lower cardiovascular (n = 340) and all-cause mortality (n = 1159) as well as a lower risk of heart failure (n = 177), stroke (n = 318) and myocardial infarction (n = 452). Smoking, diabetes and high alcohol consumption were associated with lower levels of SCF. Single nucleotide polymorphisms in the gene region encoding PDX1 C-terminal inhibiting factor 1 (PCIF1) and matrix metalloproteinase-9 were associated with plasma SCF levels. The highest SCF quartile remained independently associated with a lower risk of a lower risk of cardiovascular [hazard ratio and 95% confidence interval 0.59 (0.43-0.81)] and all-cause mortality [0.68 (0.57-0.81)], heart failure [0.50 (0.31-0.80)] and stroke [0.66 (0.47-0.92)], but not with MI [0.96 (0.72-1.27)] as compared with the lowest quartile when adjusting for traditional cardiovascular risk factors in Cox proportional hazard regression models. CONCLUSIONS: This prospective population-based study demonstrates that subjects with high levels of SCF have a lower risk of cardiovascular events and death. The findings provide clinical support for a protective role of SCF in maintaining cardiovascular integrity.

Decreased levels of stem cell factor in subjects with incident coronary events.

BACKGROUND: It has been proposed that vascular progenitor cells play an important role in vascular repair, but their possible clinical importance in cardiovascular disease has not been fully characterized. Vascular endothelial growth factor A, placental growth factor and stem cell factor (SCF) are three growth factors that are important in recruiting vascular progenitor cells. In this study, we investigated the association between the plasma levels of these growth factors and incident coronary events (CEs). METHODS: Levels of the three growth factors were measured using the proximity extension assay technique in baseline plasma samples from 384 subjects with a first CE (mean follow-up 14.0 ± 4.3 years) and 409 event-free control subjects matched by sex and age, as well as in homogenates from 201 endarterectomy specimens. RESULTS: After controlling for known cardiovascular disease risk factors in a Cox regression model, subjects in the lowest SCF tertile had a hazard ratio of 1.70 (95% confidence interval 1.14-2.54) compared with subjects in the highest SCF tertile. Lower SCF levels were also associated with more severe carotid disease, less fibrous atherosclerotic plaques and an increased incidence of heart failure. Expression of the SCF receptor c-kit was demonstrated in the subendothelial layer and fibrous cap of human atherosclerotic plaques. Smokers and subjects with diabetes had decreased levels of SCF compared with control subjects. CONCLUSION: To our knowledge, this is the first clinical study to provide evidence to support a key role for SCF and progenitor cells in vascular repair. We suggest that the SCF-c-kit pathway may be a promising biomarker and therapeutic target in cardiovascular disease.

High levels of IgM against methylglyoxal-modified apolipoprotein B100 are associated with less coronary artery calcification in patients with type 2 diabetes.

OBJECTIVE: Advanced glycation end products (AGE) have been implicated in diabetic vascular complications through activation of pro-inflammatory genes. AGE-modified proteins are also targeted by the immune system resulting in the generation of AGE-specific autoantibodies, but the association of these immune responses with diabetic vasculopathy remains to be fully elucidated. The aim of this study was to determine whether antibodies against apolipoprotein B100 modified by methylglyoxal (MGO-apoB100) are associated with coronary atherosclerosis in patients with type 2 diabetes. METHODS: We measured antibodies against MGO-apoB100 in plasma from 497 type 2 diabetic patients without clinical signs of cardiovascular disease. Severity of coronary disease was assessed as coronary artery calcium (CAC) imaging. Immunoglobulin (Ig)M and IgG levels recognizing MGO-apoB100 were determined by enzyme-linked immunosorbent assay. RESULTS: Anti-MGO-apoB100 IgM antibody levels were higher in subjects with a low to moderate CAC score (≤400 Agatston units) than in subjects with a high score (>400 Agatston units; 136.8±4.4 vs. 101.6± 7.4 arbitrary units (AU), P<0.0001) and in subjects demonstrating no progression of CAC during 30 months of follow-up (136.4±5.7 vs. 113.9 ± 6.2 AU in subjects with progression, P<0.0001). Subjects with a family history of premature myocardial infarction had lower levels of anti-MGO-apoB100 IgM. Female subjects had higher levels of anti-MGO-apoB100 antibodies and lower CAC than men. Accordingly, high levels of IgM against MGO-apoB100 are associated with less severe and a lower risk of progression of coronary disease in subjects with type 2 diabetes. CONCLUSIONS: Although conclusions regarding causal relationships based on epidemiological observations need to be made with caution, our findings suggest the possibility that anti-MGO-apoB100 IgM may be protective in diabetic vasculopathy.

Improved methodology for identifying the teratogenic potential in early drug development of hERG channel blocking drugs.

Drugs blocking the potassium current IKr of the heart (via hERG channel-inhibition) have the potential to cause hypoxia-related teratogenic effects. However, this activity may be missed in conventional teratology studies because repeat dosing may cause resorptions. The aim of the present study was to investigate an alternative protocol to reveal the teratogenic potential of IKr-blocking drugs. The IKr blocker astemizole, given as a single dose (80 mg/kg) on gestation day (GD) 13 to pregnant rats caused digital defects. In whole rat embryo culture (2h) on GD 13, astemizole caused a decrease in embryonic heart rate at 20 nM, and arrhythmias at 200-400 nM. Cetirizine, without IKr-blocking properties, did not affect the rat embryonic heart in vitro. The present study shows that single dose testing on sensitive days of development, together with whole embryo culture, can be a useful methodology to better characterize the teratogenic potential of IKr-blocking drugs.

Associations between autoantibodies against apolipoprotein B-100 peptides and vascular complications in patients with type 2 diabetes.

AIMS/HYPOTHESIS: Oxidation of LDL in the arterial extracellular matrix is a key event in the development of atherosclerosis and autoantibodies against oxidised LDL antigens reflect disease severity and the risk of developing acute cardiovascular events. Since type 2 diabetes is associated with increased oxidative stress, we tested the hypothesis that autoantibodies against oxidised LDL antigens are biomarkers for vascular complications in diabetes. METHODS: We studied 497 patients with type 2 diabetes without clinical signs of coronary heart disease. Oxidised LDL autoantibodies were determined by ELISA detecting IgG and IgM specific for native and malondialdehyde (MDA)-modified apolipoprotein B-100 peptides p45 and p210. The severity of coronary disease was assessed as the coronary artery calcium score. RESULTS: Patients affected by retinopathy had significantly higher levels of IgG against MDA-p45 and MDA-p210. In contrast, high levels of autoantibodies against the corresponding native peptides were associated with less coronary calcification and a lower risk of progression of coronary disease. CONCLUSIONS/INTERPRETATION: Our observations suggest that LDL oxidation is involved in the pathogenesis of diabetic retinopathy and that autoantibodies against apolipoprotein B peptides may act as biomarkers for both micro- and macrovascular complications in diabetes.

Immune responses against fibronectin modified by lipoprotein oxidation and their association with cardiovascular disease.

OBJECTIVES: Accumulation and subsequent oxidation of LDL in the arterial wall are considered as key events in the development of atherosclerosis. We have investigated the possibility that LDL oxidation results in release of aldehydes that modify surrounding matrix proteins and that this may target immune responses against the plaque extracellular matrix and modulate the disease progression. RESULTS: Using custom-made ELISAs we demonstrate that human plasma contains autoantibodies against aldehyde-modified fibronectin (FN) and to a lesser extent also other extracellular matrix proteins including collagen type I, type III, and tenascin-C. Immunohistochemistry and western blot analysis showed that aldehyde-modified FN is present in human atherosclerotic plaques and that aldehydes generated by oxidation of LDL formed adducts with FN in vitro. We also demonstrate that aldehyde-modification of FN results in a loss of its ability to promote basal secretion of cytokines and growth factors from cultured macrophages without affecting the ability of the cells to respond to stimulation with LPS. A prospective clinical study demonstrated that subjects that subsequently developed acute myocardial infarction or sudden cardiac death had lower baseline levels of autoantibodies against aldehyde-modified FN than matched controls. CONCLUSIONS: These observations demonstrate that oxidation of LDL in the arterial wall may lead to aldehyde-modification of surrounding extracellular matrix proteins and that these modifications may affect macrophage function and activate autoimmune responses of pathophysiological importance for the development of atherosclerosis.

Combining intensity, edge and shape information for 2D and 3D segmentation of cell nuclei in tissue sections.

We present a region-based segmentation method in which seeds representing both object and background pixels are created by combining morphological filtering of both the original image and the gradient magnitude of the image. The seeds are then used as starting points for watershed segmentation of the gradient magnitude image. The fully automatic seeding is done in a generous fashion, so that at least one seed will be set in each foreground object. If more than one seed is placed in a single object, the watershed segmentation will lead to an initial over-segmentation, i.e. a boundary is created where there is no strong edge. Thus, the result of the initial segmentation is further refined by merging based on the gradient magnitude along the boundary separating neighbouring objects. This step also makes it easy to remove objects with poor contrast. As a final step, clusters of nuclei are separated, based on the shape of the cluster. The number of input parameters to the full segmentation procedure is only five. These parameters can be set manually using a test image and thereafter be used on a large number of images created under similar imaging conditions. This automated system was verified by comparison with manual counts from the same image fields. About 90% correct segmentation was achieved for two- as well as three-dimensional images.

Automatic registration and error detection of multiple slices using landmarks.

OBJECTIVES: When analysing the 3D structure of tissue, serial sectioning and staining of the resulting slices is sometimes the preferred option. This leads to severe registration problems. In this paper, a method for automatic registration and error detection of slices using landmark needles has been developed. A cost function takes some parameters from the current state of the problem to be solved as input and gives a quality of the current solution as output. The cost function used in this paper, is based on a model of the slices and the landmark needles. The method has been used to register slices of prostates in order to create 3D computer models. Manual registration of the same prostates has been undertaken and compared with the results from the algorithm. METHODS: Prostates from sixteen men who underwent radical prostatectomy were formalin fixed with landmark needles, sliced and the slices were computer reconstructed. The cost function takes rotation and translation for each prostate slice, as well as slope and offset for each landmark needle as input. The current quality of fit of the model, using the input parameters given, is returned. The function takes the built-in instability of the model into account. The method uses a standard algorithm to optimize the prostate slice positions. To verify the result, s standard method in statistics was used. RESULTS: The methods were evaluated for 16 prostates. When testing blindly, a physician could not determine whether the registration shown to him were created by the automated method described in this paper, or manually by an expert, except in one out of 16 cases. Visual inspection and analysis of the outlier confirmed that the input data had been deformed. The automatic detection of erroneous slices marked a few slices, including the outlier, as suspicious. CONCLUSIONS: The model based registration performs better than traditional simple slice-wise registration. In the case of prostate slice registration, other aspects, such as the physical slicing method used, may be more important to the final result than the selection of registration method to use.

The amino-terminal part of PRELP binds to heparin and heparan sulfate.

PRELP (proline, arginine-rich end leucine-rich repeat protein) is an extracellular matrix leucine-rich repeat protein. The amino-terminal region of PRELP differs from that of other leucine-rich repeat proteins in containing a high number of proline and arginine residues. The clustered proline and basic residues are conserved in rat, bovine, and human PRELP. Although the function of PRELP is not yet known, the clustered arginine residues suggest a heparan sulfate/heparin-binding capacity. We show here that PRELP indeed binds heparin and heparan sulfate. Truncated PRELP without the amino-terminal region does not bind heparin. The dissociation constant for the interaction of PRELP with heparin was determined by an in solution binding assay and by surface plasmon resonance analysis to be in the range of 10-30 nm. A 6-mer heparin oligosaccharide was the smallest size showing binding to PRELP. The binding increased with increasing length up to an 18-mer and depended on the degree of sulfation of heparin as well as heparan sulfate. Sulfate groups at all positions were shown to be of importance for the binding. Fibroblasts bind PRELP, and this interaction is inhibited with heparin, suggesting a function for PRELP as a linker between the matrix and cell surface proteoglycans.

A detailed analysis of cyclin A accumulation at the G(1)/S border in normal and transformed cells.

The temporal relationship between cyclin A accumulation and the onset of DNA replication was analyzed in detail. Five untransformed and nine transformed asynchronously growing cell cultures were investigated using a triple immunofluorescence staining protocol combined with computerized evaluation of staining intensities in individual cells. The simultaneous staining of BrdU, cyclin A, and cyclin E made it possible to determine the cell cycle position of each cell investigated. Cells at the G(1)/S border were identified on the basis of cyclin E content and were further analyzed with respect to cyclin A and BrdU content. A method was developed to calculate objective thresholds defining the highest staining intensity found in the negative cells in the population. Using the thresholds we could distinguish cells with minute amounts of cyclin A and BrdU from truly negative cells. We show that the onset of cyclin A accumulation and the start of DNA replication occurs at the same time, or deviating by a few minutes at the most. We also show that cyclin A accumulates continuously during S. This study clearly demonstrates that nuclear cyclin A can be used as a reliable marker for the S and G(2) phases in both normal and transformed interphase cells.

Paraffin section storage and immunohistochemistry. Effects of time, temperature, fixation, and retrieval protocol with emphasis on p53 protein and MIB1 antigen.

It has been observed that immunoreactivity in paraffin sections decreased during storage. In this study, stored paraffin sections from both biopsy material and cultured cells were assessed for changes in immunoreactivity, using color-based image analysis to quantitate extent and intensity of the stainings. For seven of the 11 antibodies studied, storage at 20 degrees C for 16 weeks reduced the extent of immunostaining compared with that of freshly cut sections. Furthermore, increased storage temperatures resulted in a progressive loss of immunoreactivity. After 2 weeks of storage, at both 4 degrees C and 20 degrees C, p53 protein- and MIB1-antigen expression was significantly reduced regarding extent and intensity. The extent of the immunoreactivity reduced more for p53 protein than for MIB1 antigen, but the intensity did not. Boric acid was used for antigen retrieval on sections stored for 12 weeks at 20 degrees C. For both p53 protein and MIB1 antigen, this resulted in an extent and intensity of immunostaining equal to or higher than (MIB1) that obtained in freshly cut sections, using citrate buffer. Staining of cultured cells confirmed the results from biopsy material on the influence of storage temperature. Fixation time only marginally influenced the storage-related decrease in immunoreactivity. In conclusion, storage of paraffin sections leads to a varying degree of decreased immunoreactivity for several antibodies. The degree is at least partly dependent on storage time and temperature but not fixation time. However, this may be compensated for by optimizing the antigen retrieval protocol.

Cultured human fibroblasts in agarose gel as a multi-functional control for immunohistochemistry. Standardization Of Ki67 (MIB1) assessment in routinely processed urinary bladder carcinoma tissue.

Immunohistochemistry (IHC) in clinical practice is hampered by lack of standardization and by subjectivity in interpretation and quantitation. This study aimed to develop a control system for IHC in routinely fixed and histoprocessed tissues. Such a system should be easy to handle in clinical practice and should reflect variations in fixation time, section thickness, section storage conditions, and staining protocols. In addition, in image analysis quantitation of immunostained tissues, when using classifiers computed on IHC-control images, the control system should be very stable. Cultured human fibroblasts were suspended in agarose, transferred into a length of tubing and stored at 4 degrees C. Three pieces of the cellgel control were separately fixed, histoprocessed, and paraffin-embedded as external controls. One piece was prepared together with each of 18 bladder carcinoma biopsies as internal controls. Slides with sections from the biopsy and all types of cellgel controls were stored at different temperatures and then stained using three different IHC protocols. The fibroblasts were homogeneously distributed in the agarose gel. Variation in section thickness did not influence immunostaining as evaluated by the MIB1 labelling index (MIB1 LI). The external controls decreased notably in MIB1 LI with increased fixation time. This was not seen in the 18 internal controls that were each fixed with a fresh biopsy. However, section storage and immunostaining conditions influenced the MIB1 expression equally in all control types and to a similar degree to the biopsies. Furthermore, colour-based image analysis quantitation of MIB1 LI in biopsies proved stable and independent of the control type used to compute the classifier.

Modeling prostate cancer distributions.

OBJECTIVES: To use computer-assisted three-dimensional (3D) reconstruction to study cancer distribution in the prostate. The distributions have been determined using data from 81 prostates surgically removed because of cancer. METHODS: The pattern of distribution was determined by reshaping (morphing) all prostates in the same size category (small, medium, large) into the same shape and by overlayering the 3D distribution of all cancers into a "unit prostate." Also, the pattern of 3D distribution was defined for small, medium, and large volume cancers. RESULTS: The study yielded a clear, visual "scatter diagram" representation of the statistics of prostate cancer distribution. It confirmed previous general knowledge: small cancers are mainly found in the lower one third and principally in the dorsolateral part of the gland. CONCLUSIONS: This report is part of the investigations preliminary to establishing an optimal protocol for needle biopsy of prostate cancer.

Automatic quantification of microvessel density in urinary bladder carcinoma.

Seventy-three TUR-T biopsies from bladder carcinoma were evaluated regarding microvessel density, defined as microvessel number (nMVD) and cross-section endothelial cell area (aMVD). A semi-automatic and a newly developed, automatic image analysis technique were applied in immunostainings, performed according to an optimized staining protocol. In 12 cases a comparison of biopsy material and the corresponding cystectomy specimen were tested, showing a good correlation in 11 of 12 cases (92%). The techniques proved reproducible for both nMVD and aMVD quantifications related to total tumour area. However, the automatic method was dependent on high immunostaining quality. Simultaneous, semi-automatic quantification of microvessels, stroma and epithelial fraction resulted in a decreased reproducibility. Quantification in ten images, selected in a descending order of MVD by subjective visual judgement, showed a poor observer capacity to estimate and rank MVD. Based on our results we propose quantification of MVD related to one tissue compartment. When staining quality is of high standard, automatic quantification is applicable, which facilitates quantification of multiple areas and thus, should minimize selection variability.

Biopsy protocol stability in a three-dimensional model of prostate cancer: changes in cancer yield after adjustment of biopsy positions.

OBJECTIVES: Transrectal ultrasound-guided prostate biopsies are often taken according to a systematic, standardized schedule. The diagnostic stability of this system was evaluated by moving the biopsies in a three-dimensional (3D) model. METHODS: A computerized 3D reconstruction was made from each of 75 radical prostatectomy specimens. Simulated core biopsies imitated a standardized 10-biopsy protocol, including sextant biopsies. In total, 30,000 biopsies were generated by moving the standardized biopsies 1, 2, 3, and 4 mm (parallel needle shifts) or 5 degrees, 10 degrees, 15 degrees, and 20 degrees(rotation of the needle tip) in a random direction. RESULTS: The diagnosis of the individual biopsy changed from cancer to benign or vice versa in 4.9% to 1 5.7% after 1 to 4-mm parallel needle shifts and 2.0% to 7.5% after 5 degrees to 20 degrees rotations. The corresponding figures for the final diagnosis of the 10-biopsy set were 0.8% to 9.6% and 0.5% to 3.2%. Transition zone biopsies containing cancer changed to benign more often than the other biopsies (P <0.001). Parallel needle shifts of 2 mm changed the diagnosis more often than the 15 degrees rotation (9.4% and 5.9%, respectively, P <0.001), although conveying the same overall needle shift. CONCLUSIONS: The cancer yield of prostate biopsies is influenced even by small changes in needle positions. The transition zone biopsies are most likely to change from cancer to benign when moved. Changing the insertion point of the needle has a higher impact on cancer yield than rotating the tip.