Prognostic markers for colorectal cancer: estimating ploidy and stroma.

Background: We report here the prognostic value of ploidy and digital tumour-stromal morphometric analyses using material from 2624 patients with early stage colorectal cancer (CRC). Patients and methods: DNA content (ploidy) and stroma-tumour fraction were estimated using automated digital imaging systems and DNA was extracted from sections of formalin-fixed paraffin-embedded (FFPE) tissue for analysis of microsatellite instability. Samples were available from 1092 patients recruited to the QUASAR 2 trial and two large observational series (Gloucester, n = 954; Oslo University Hospital, n = 578). Resultant biomarkers were analysed for prognostic impact using 5-year cancer-specific survival (CSS) as the clinical end point. Results: Ploidy and stroma-tumour fraction were significantly prognostic in a multivariate model adjusted for age, adjuvant treatment, and pathological T-stage in stage II patients, and the combination of ploidy and stroma-tumour fraction was found to stratify these patients into three clinically useful groups; 5-year CSS 90% versus 83% versus 73% [hazard ratio (HR) = 1.77 (95% confidence interval (95% CI): 1.13-2.77) and HR = 2.95 (95% CI: 1.73-5.03), P < 0.001]. Conclusion: A novel biomarker, combining estimates of ploidy and stroma-tumour fraction, sampled from FFPE tissue, identifies stage II CRC patients with low, intermediate or high risk of CRC disease specific death, and can reliably stratify clinically relevant patient sub-populations with differential risks of tumour recurrence and may support choice of adjuvant therapy for these individuals.

Prognostic impact of genomic instability in colorectal cancer.

BACKGROUND: The prognostic impact of an indication of chromosomal instability (CIN) is evaluated in a consecutive series of 952 colorectal cancer patients treated at Aker University Hospital, Norway, during 1993-2003. Microsatellite instability (MSI) in this case series has recently been reported and made it possible to find the co-occurrence and compare the prognostic significance of CIN and MSI. METHODS: Data sets for overall survival (OS; n=855) and time to recurrence (TTR; n=579) were studied. To reveal CIN we used automated image cytometry (ICM). Non-diploid histograms were taken as indicative of the presence of CIN. PCR-based measures of MSI in this material have already been described. RESULTS: As with MSI, CIN was found to be an independent predictor of early relapse and death among stage II patients (TTR: n=278: HR 2.19 (95% CI: 1.35-3.55), P=0.002). Of the MSI tumours (16%), 71% were found to be DNA diploid, 21% were DNA tetraploid and 8% were DNA aneuploid. Among microsatellite stable tumours, 24% were DNA diploid, 15% were DNA tetraploid and 61% were DNA aneuploid. CONCLUSION: For patients presenting with stage II disease, genomic instability as detected by DNA image cytometry has the potential to provide a useful biomarker for relapse and cancer-related death following surgery with curative intent.

Microsatellite instability has a positive prognostic impact on stage II colorectal cancer after complete resection: results from a large, consecutive Norwegian series.

BACKGROUND: Microsatellite instability (MSI) was suggested as a marker for good prognosis in colorectal cancer in 1993 and a systematic review from 2005 and a meta-analysis from 2010 support the initial observation. We here assess the prognostic impact and prevalence of MSI in different stages in a consecutive, population-based series from a single hospital in Oslo, Norway. PATIENTS AND METHODS: Of 1274 patients, 952 underwent major resection of which 805 were included in analyses of MSI prevalence and 613 with complete resection in analyses of outcome. Formalin-fixed tumor tissue was used for PCR-based MSI analyses. RESULTS: The overall prevalence of MSI was 14%, highest in females (19%) and in proximal colon cancer (29%). Five-year relapse-free survival (5-year RFS) was 67% and 55% (P = 0.030) in patients with MSI and MSS tumors, respectively, with the hazard ratio (HR) equal to 1.60 (P = 0.045) in multivariate analysis. The improved outcome was confined to stage II patients who had 5-year RFS of 74% and 56% respectively (P = 0.010), HR = 2.02 (P = 0.040). Examination of 12 or more lymph nodes was significantly associated with proximal tumor location (P < 0.001). CONCLUSIONS: MSI has an independent positive prognostic impact on stage II colorectal cancer patients after complete resection.

The localization of sudanophilic and fibrous plaques in the main left coronary bifurcation.

We have studied the localization of early sudanophilic and fibrous plaques in the main stem and proximal branches of opened and stained left coronary arteries. The distribution of lesions was separately assessed by a computer technique. The early sudanophilic lesions develop in the proximal portion of the coronary artery branches and subsequent lesions spread distally and appear first and most prominently in the left anterior descending branch. Around the bifurcation both sudanophilic and fibrous plaques had a distinct spatial distribution with peak incidence on the outer walls. In contrast, the flow divider and the inner walls downstream from it were relatively free of disease.

Distribution pattern of sudanophilic plaques in the descending thoracic and proximal abdominal human aorta.

The localization of sudanophilic plaques around the intercostal ostia and the origin of the superior mesenteric and coeliac artery was examined in 38 human aortae. The perimeter of the opened aorta and the outline of each sudanophilic plaque were digitized and computerized. By means of a special program the results were displayed as progressive contour lines connecting points with an equal frequency of plaques. This study shows that the sudanophilic plaques have a distinct spatial distribution both in the thoracic and abdominal aorta. Around the intercostal ostia peak frequency of plaques is found just proximal to the lower lip of the ostia. In the bifurcation of both the superior mesenteric and coeliac artery the plaques are present on the outer walls while the inner walls and the apex itself are free of plaques.

Localization of atherosclerotic lesions in the bifurcation of the main left coronary artery.

The detailed distribution of atherosclerotic lesions in the branching region of the left main coronary artery in man was studied. Tissue cubes of the hearts, containing the left coronary arteries, were removed, and 1.5-mm-thick parallel and consecutive slices were cut perpendicular to the main coronary artery. Histological sections of the slices were stained and photographed. Drawings of the cross-sections of the arteries, including information about thickness of the intima and borders of the atherosclerotic lesions, were fed into a computer, scaled and analyzed. Casts were made of the left main coronary bifurcation from additional hearts. The curvature of the arteries and the angles of the bifurcation were measured. Atherosclerotic lesions have a distinct pattern with a high frequency on the outer walls of the bifurcation and at the inner curvature downstream from the bifurcation. The extent of intimal thickening and the occurrence of atherosclerotic lesions were mostly in agreement.

Histopathology of arterial lesions in LPA transgenic mice on cholesterol-enriched chow.

The aortic root from 21 LPA transgenic mice and 18 control litter mates on cholesterol enriched chow were studied histologically for the presence of atherosclerotic lesions. Serial sections were cut and the total area of the lesions was measured by use of computerised image analysis. Lipid staining lesions were found in 17 aortas of the transgenic mice and were five times more common than in the controls. Foam cell lesions were the only type of lesion in 12 of the aortas from transgenic animals, while five animals had developed fibrofatty lesions. Immunostaining revealed monocytes/macrophages on the endothelial surface, and in the subendothelial space of foam cell lesions. In fibrofatty lesions, spindle shaped cells formed a cap around the lipid core. This study supports the view that transgenic mice expressing human apolipoprotein (a) on a high fat and cholesterol diet, are more susceptible to aortic lesions than control mice and develop early atherosclerotic lesions comparable to lesions in man. Aminoguanidin in the drinking water had no effect on the aortic lesions, but lesion size was significantly, negatively correlated with plasma glucose concentration.

Aminoethyl-isothiourea inhibits the increase in plasma endothelin-1 caused by serogroup A streptococci and prolongs survival in rat peritoneal sepsis.

To elucidate the possible roles of nitric oxide (NO), endothelin-1 (ET-1), and reactive oxygen species (ROS) in the pathophysiology of serogroup A streptococcal (GAS) peritoneal sepsis, we investigated the effects of aminoethylisothiourea (AE-ITU), an inducible NO synthase (iNOS) inhibitor, and a ROS scavenger, and the ET-1 receptor antagonist bosentan. In rats, live GAS inocula, 3 x 10(8) and 1 x 10(9) cfu/kg, entailed a 24-h mortality of 10% and 90%, respectively. GAS caused increases in tissue iNOS activity (9 h), in serum nitrite/nitrate (9-24 h), and in intracellular leukocyte ROS levels (3-6 h). These changes were all prevented by the pre-treatment with AE-ITU. A novel finding was that AE-ITU also prevented the GAS-induced marked increase in plasma ET-1 at 6 h. Short-term (7-h) survival was improved by both AE-ITU and by bosentan. The mechanism(s) for the beneficial effects of AE-ITU may possibly be a combined mode of action; iNOS inhibition, ROS scavenging, and inhibition of the increase in plasma ET-1 caused by GAS.

Carvedilol reduces ischaemic skeletal muscle necrosis.

Carvedilol is an alpha1 and nonselective beta-adrenergic receptor antagonist with antioxidative properties known to protect against reperfusion injury in the heart, brain, and kidneys. The aim of this study was to test the hypothesis that carvedilol improves postischaemic reperfusion and tissue survival in skeletal muscle. Sixteen Wistar rats underwent tourniquet ischaemia of the left hindlimb for 3 hours and 15 minutes at 27 degrees C. Single-fiber laser Doppler probes were inserted in the left and right anterior tibial muscles, and microvascular perfusion was measured until 2 hours after removal of the tourniquet. Perfusion indices for each 15-minute interval were calculated for the left hindlimb (tourniquet ischaemia) by dividing the postischaemic by the pre-ischaemic laser Doppler flowmetry values, and the geometrical areas under the curves representing a plot of perfusion index relative to time, measured in arbitrary units, were compared. Laser Doppler flowmetry values for the right anterior tibial muscle were compared. Tissue damage was measured by histomorphometry of necrotic areas and no-reflow zones in cross sections from the anterior tibial muscle 72 hours after ischaemia. Neutrophils were counted in the same sections. The treatment group received 1 mg carvedilol/kg body weight before ischaemia and 1 mg/kg immediately before removal of the tourniquets. The areas under the curves representing the plot of perfusion index relative to time were larger for the rats treated with carvedilol: 9.5 compared with 3.0 arbitrary units (p = 0.0003). Treatment did not change the laser Doppler flowmetry values for the right hindlimbs. The histomorphometric areas of necrosis in cross sections from the muscles were reduced from 88% (38-96%) in the control animals to 41% (7-85%) in those treated with carvedilol (p = 0.01), and the area of no-reflow was reduced from 20% (2-52%) to 0% (0-7%) (p = 0.006). The number of neutrophils did not differ between groups. The study supports the hypothesis that carvedilol improves early reperfusion and protects skeletal muscle subjected to 3 hours and 15 minutes of ischaemia.

Endothelin-1 is upregulated during skeletal muscle ischemia and reperfusion.

The aim of the present study was to test the hypothesis that the vasoconstrictive peptide endothelin-1 is upregulated in ischemia and reperfusion in skeletal muscle. Sixty-eight Wistar rats were included in the series: 12 served as controls that did not undergo the procedure, 16 underwent sham operations, and 40 were subjected to a modified tourniquet ischemia for 3 hours and 20 minutes. Of the 40 rats, 16 were killed at the end of the ischemic period, 16 underwent reperfusion for 2 hours, and eight underwent reperfusion for 72 hours. Areas of necrosis were measured by morphometry in hematoxylin and eosin-stained cross sections of the anterior tibial muscles that had been reperfused for 72 hours. Sections from the controls, the muscles that had not been reperfused, and the reperfused muscles were immunostained for endothelin-1. Serum endothelin-1 levels in blood samples from the aorta were determined with a commercial enzyme immunoassay kit. The anterior tibial muscle was harvested for preproendothelin-1 mRNA analysis with RNase protection assay. The hematoxylin and eosin-stained sections showed extensive necrosis with an acellular core of no reperfusion. The muscular core demonstrated weak immunostaining for endothelin-1 in all sections, a subfascial narrow brim of fibers showed enhanced immunoreactivity at the end of ischemia, and all fibers outside the core stained by 2 hours after the start of reperfusion. After 72 hours of reperfusion, the fibers outside the core stained positive in a checkerboard-like pattern. There were no differences in serum endothelin-1 levels between the groups. Preproendothelin-1 mRNA analysis with RNase protection assay showed 2-fold upregulation at the end of ischemia and 4-fold upregulation after 2 hours of reperfusion (p = 0.001). This study supports the hypothesis that both ischemia and reperfusion upregulate endothelin-1 in skeletal muscle.

Transventricular non-transmural laser treatment of hypoperfused porcine myocardium acutely reduces left ventricular contractile function.

OBJECTIVE: Creation of non-transmural myocardial channels by lasers transmitted through endovascular fiberoptics is a novel therapeutic option in the management of patients with coronary artery disease. The acute effect of transventricular laser treatment (TvL) on coronary blood flow, myocardial metabolism and left ventricular function are not well established. METHODS: In five anesthetized pigs, flow in the proximal left anterior descending coronary artery (LAD) was reduced and maintained at 70% of baseline. A venous shunt had previously been established draining the hypoperfused region. At 30 min of ischemia, non-transmural myocardial channels were created through the endocardium using a Ho:YAG laser. We measured (a) left ventricular, central venous and arterial pressures, (b) ascending aortic, LAD and coronary venous blood flows, as well as (c) lactate concentration and blood gases in arterial and coronary venous blood, prior to ischemia (baseline), before and 30 min after TvL. Data (given as mean +/- SD) were analyzed with repeated measures ANOVA. RESULTS: Reduction of LAD blood flow resulted in reduced regional coronary venous blood flow and myocardial oxygen consumption, conversion of regional myocardial lactate uptake to release and adaptation of left ventricular contractility to a lower level. Following transventricular laser, the peak left ventricular systolic pressure declined from 86 +/- 12 to 77 +/- 11 mmHg (P < 0.05), its maximal first positive derivative (LV dP/dt) declined from 900 +/- 221 to 763 +/- 127 mmHg/s (P < 0.05) and the stroke volume decreased from 19.2 +/- 4.1 to 16.4 +/- 5.4 ml (P < 0.05). The changes in regional coronary venous flow, myocardial oxygen consumption and myocardial lactate release after TvL were not significant compared to before TvL. Significant intramural hematomas and tissue destruction were found around the channels at autopsy and by histologic examination. CONCLUSION: Transventricular laser treatment of hypoperfused myocardium decreased left ventricular contractility in the acute phase, possibly due to development of perichannel hematomas and disruption of the wall architecture. In addition, TvL did not alter the regional myocardial oxygen supply/demand balance. These results call for caution in the treatment of patients with coronary artery disease by transventricular Ho-YAG laser when there is significant impairment of the left ventricular contractile function.

Morphometry of benign prostatic hyperplasia during androgen suppressive therapy. Relationships among epithelial content, PSA density, and clinical outcome.

We performed light microscopic morphometry on prostate biopsies of 41 patients who underwent androgen suppressive therapy for 24 weeks with the luteinizing hormone releasing hormone (LHRH) agonist leuprolide depot for symptomatic benign prostatic hyperplasia (BPH). Before treatment started, the prostates consisted of 88.4% stroma, 9.0% epithelium and 2.6% glandular lumen. After completion of therapy, the percentages were 94.7, 3.0 and 2.3, respectively. The absolute reductions of volume were 27% for stroma, 77% for epithelium and 40% for lumen. Correlations between pretreatment parameters (epithelial content, stromal epithelial ratio and prostate specific antigen density (PSAD)) and the clinical outcome parameters (prostate volume reduction, improvement in maximum flow rate, reduction of symptom score and reduction in outflow resistance) were not statistically significant, indicating that the histological composition of BPH tissue or PSAD are poor parameters to predict differences in response for individual patients during hormonal therapy for BPH. By comparing morphometry results of the core biopsies with morphometry performed on corresponding TURP tissue, it appeared that the lumen content of the biopsies was somewhat underestimated.

Endoscopic snare resection followed by laser ablation in the treatment of large, sessile rectal adenomas.

BACKGROUND AND AIMS: Large sessile rectal adenomas can be difficult to eradicate, and different treatment modalities are available. The aim of this study was to evaluate outcome after endoscopic snare resection followed by Nd:YAG laser ablation. MATERIAL AND METHODS: Over a 10-year period 92 of 99 (93%) patients were registered prospectively and attended follow-up examinations with endoscopy and biopsies. RESULTS: Fifty-four (59%) men and 38 (41%) women were included; 67 patients (73%) had high grade (severe) intraepithelial dysplasia or intramucosal neoplasia. The adenomas ranged from 2-9 cm (median 4 cm) in diameter, and were located 2-15 cm (median 5 cm) from the anal verge. A median of two (range 1-6) piecemeal snare resection sessions and a median of one (range 1-7) laser treatments were performed for each patient. Complete eradication was achieved in 86 patients (93%). Over a median follow-up period of 26 months, 20/86 (23%) suffered local recurrence, eight of whom were given a second laser treatment without developing further recurrence. In five of eight frail patients considered unsuitable for more radical treatment, repeated laser treatment was effective in keeping the adenoma small and symptoms at a minimum. As a whole the treatment was successful in 74/92 (80%) and partially successful in 5/92 (5%) of the patients. CONCLUSIONS: Snare resection followed by laser ablation is safe and still has a place in the treatment of old, frail patients with large rectal adenomas. However, there is a risk of missing an infiltrating carcinoma, and other treatment options are preferable in fit patients.

Development of intimal thickening of coronary arteries over the lifetime of Atlantic salmon, Salmo salar L., fed different lipid sources.

The objective of the present study was to investigate the development of intimal changes of coronary arteries over the lifetime of farmed Atlantic salmon, Salmo salar L., fed either a 100% fish oil or a 100% vegetable oil blend. The study was performed as a randomized observer blinded controlled trial with parallel group design. At the start of the project, the fish were divided in two groups and sampled at five different time points throughout their life span. The total study sample consisted of 259 healthy fish. Serial sections were taken from the coronary artery lying on the bulbus arteriosus for histopathological evaluation and for area measurements using semi-quantitative and quantitative methods. The earliest onset of vascular changes was detected in fish from both groups in the freshwater stage prior to smoltification. The mean range lesion (MRL), used to describe the severity of the lesions observed, increased significantly for both groups from sea transfer throughout the study period. Comparison of the two groups based on the overall material corrected for time of sampling did not show any difference (P = 0.20) between the two groups with regard to MRL. The percentage lumen loss (PLL) measured by a quantitative method and used as a measure to indicate lesion severity showed an incremental, non-significant increase from week 72 to week 92 and further to week 115 in both diet groups during the seawater phase. Comparison of the groups corrected for time of sampling indicated a difference of PLL in favour of VO (P = 0.02). Heart weight, body weight and body length were all positively and significantly correlated to Log MRL. The partial correlation analysis indicated that heart weight was the most dominant variable in the set. Early vascular changes were found in the major bifurcation of the coronary artery at the apex and beyond the flow divider into the daughter branches. The latter represented the dominant changes and were found throughout the entire lifecycle of the fish. Increasing in size over time they formed pads or cushions that were regularly located close to the outer walls of the bifurcation. The origin of the cells forming the intimal thickening has not been conclusively determined, but immunohistochemical findings indicate a smooth muscle cell origin, possibly of a myointimal type. Our findings suggest there is no correlation between diet and intimal changes. The severity of the changes, MRL and PLL, of the coronary vessels correlate with heart weight and fish weight growth and growth rate and mechanical factors are implicated in intimal development, but rather than being induced by external injury due to the location of the coronaries, haemodynamic factors and low shear stress are proposed as the main mechanism behind these changes.

Influence of dietary lipid composition on cardiac pathology in farmed Atlantic salmon, Salmo salar L.

The present study investigated the short-term (5 months) effect of replacing dietary marine oils with vegetable oils on the development of arteriosclerotic changes in the heart of Atlantic salmon, Salmo salar. The experiment was performed as a randomized observer-blinded and controlled trial. Farmed Atlantic salmon were randomly sampled from a study population containing 900 individuals. The salmon were divided into three groups and given diets with either 100% fish oil (Diet 1), a 50/50% mixture of fish oil and rapeseed oil (Diet 2) or 100% rapeseed oil (Diet 3). Ten sexually immature salmon from each dietary group were sampled in March and August 2002. Additionally, 47 sexually mature wild salmon were randomly collected in mid-September 2001. Serial histological sections were taken from the bulbus arteriosus and ventricle wall for histopathological evaluation of the coronary arteries and myocardium. No significant differences in mean coronary changes recorded by the main variable 'mean range lesion' (MRL) were detected between the groups in March or August. MRL increased significantly between March and August with Diet 2 (P < 0.01), was nearly significant with Diet 3 (P = 0.06) and was unchanged with Diet 1. This pattern coincided with the Diet 2 group having the highest increase in heart weight. MHC class II immunoreactive cells in the coronary changes were detected in sections from one individual in each group. Heart weight was the most dominant variable in the data set and explained linearly 15.5% of the variation in MRL. Body weight, fish length and heart weight were all significantly, positively and linearly correlated to MRL. The Diet 2 group had the highest growth rate and also exhibited a significant increase in MRL. The possible influence of diet composition on weight gain and MRL needs to be further elucidated. Increase in heart weight seems to be the dominating predictor of the appearance of MRL in Atlantic salmon. However, the present results cannot exclude the possibility that differences in fatty acid composition of fish feed can influence the development of arteriosclerotic changes in Atlantic salmon.

Localization of atherosclerotic lesions in three cerebral arterial bifurcations.

The localization of macroscopic atherosclerotic lesions have been studied in the basilar bifurcation and the left and right internal carotid bifurcations in humans. The distribution of lesions was separately assessed by a computer technique and results were displayed as contour lines. Lesions are most frequent on the outer walls of the bifurcations and on the inner curvature of the arterial bends. The material was processed for histology, and computerised morphometry was used to study the interrelationship of microscopic atheromatous lesions and intimal thickening. The percentage of the specimens with lesions around the circumference of the arteries was calculated. The peaks and troughs The peaks and troughs of histological lesions correspond exactly to the maximum and minimum contours of the macroscopic studies. Results from the microscopic examination indicate that intimal thickening and atherosclerosis are closely related processes. However, they cannot support the view that focal pad-like areas of intimal thickening are particular precursors of atheroma.

Atherosclerotic carotid disease in asymptomatic individuals: An histological study of 53 cases.

The carotid bifurcation was studied histologically in 53 consecutive autopsies from persons over 65 years of age who were asymptomatic for carotid artery disease and showed no carotid related brain infarcts. The autopsy material is considered to be representative for the population in Oslo. The area stenosis was determined morphometrically in histological sections and the cases were examined for the occurrence of atherosclerotic plaques, calcifications, plaque hemorrhages, ulcerations, and mural thrombi. About one half of the cases had more than 50% stenosis. There was increasing frequency of plaque hemorrhages, ulcerations and mural thrombi with increasing stenosis. When the stenosis exceeded 60%, most cases had small recent and old plaque hemorrhages and one half had ulcerations and mural thrombi. In addition, numerous healed ulcerations and organized thrombi were seen. It is concluded that plaque complications are frequent in cases with stenosis and that most of them apparently heal without giving rise to symptoms. The presence of such lesions in asymptomatic cases must therefore be interpreted with caution.

Is there a transitional zone between brain infarcts and the surrounding brain? A histological study.

Twenty-eight recent brain infarcts were studied histologically for the presence of zones of partial tissue destruction between the infarct border and the surrounding normal brain. Such transitional zones have been demonstrated in animals and might be suspected in humans from the broad zones of reduced blood flow that have been demonstrated around brain infarcts. Necrotic neurons were observed outside the infarct borders in 20 of 28 cases. However, the width of the peri-infarct border zone was less than 5 mm in 17 of 20 cases and between 7 and 10 mm in 2 cases. Only one section from one case showed necrotic neurons more than 20 mm from the infarct border. It is concluded that the large majority of the brain infarcts in man are sharply delimited and that the broad zones of reduced flow around the infarcts may be due to functional inactivity following partial denervation of the cortex rather than to true ischemia.

Localization of early atherosclerotic lesions in the right carotid bifurcation in humans.

The detailed localization of early atherosclerotic lesions in the right carotid bifurcation was studied in 37 carotid arteries obtained from autopsies of young persons who had suffered a violent and sudden death. Drawings of the sudanophilic lesions in the vessel wall were fed to a computer, scaled to a standardized size and shape, and added together. The results are presented as contour lines connecting points with equal frequency of early lesions. The distribution of early lesions reveals a distinct pattern: The early lesions start to develop on the outer walls of the bifurcation, while the inner walls downstream from the flow divider are left free, as are also the lateral walls. In the carotid sinus the distribution of the lesions has a U-formed pattern.