Similar insulin regulation of splanchnic FFA and VLDL-TG in men with nonalcoholic hepatic steatosis and steatohepatitis.

This study aimed to determine whether obese men with nonalcoholic fatty liver disease (NAFLD) display differences between those with simple steatosis versus steatohepatitis (NASH) in splanchnic and hepatic FFA and VLDL-triglycerides (VLDL-TG) balances. The study involved 17 obese men with biopsy-proven NAFLD (9 with NASH and 8 with simple steatosis). We used hepatic vein catheterization in combination with [3H]palmitate and [14C]VLDL-TG tracers to measure splanchnic palmitate and VLDL-TG uptake and release rates during basal and hyperinsulinemic conditions. Indocyanine green was used to measure splanchnic plasma flow. Splanchnic palmitate uptake was similar in the two groups and significantly reduced during hyperinsulinemia (NASH: 62 (48-77) versus 38 (18-58) µmol/min; simple steatosis: 62 (46-78) versus 45 (25-65) µmol/min, mean (95% CI), basal versus clamp periods, respectively, P = 0.02 time-effect). Splanchnic palmitate release was also comparable between groups and nonsignificantly diminished during hyperinsulinemia. The percent palmitate delivered to the liver originating from visceral adipose tissue lipolysis was similar and unchanged by hyperinsulinemia. Splanchnic uptake and release of VLDL-TG were similar between groups. Hyperinsulinemia suppressed VLDL-TG release (P <0.05 time-effect) in both groups. Insulin-mediated glucose disposal was similar in the two groups (P = 0.54). Obese men with NASH and simple steatosis have similar splanchnic uptake and release of FFA and VLDL-TG and a similar proportion of FFA from visceral adipose tissue lipolysis delivered to the liver. These results demonstrate that the splanchnic balances of FFA and VLDL-TG do not differ between obese men with NASH and those with simple steatosis.

Exercise increases myocardial free fatty acid oxidation in subjects with metabolic dysfunction-associated fatty liver disease.

BACKGROUND AND AIMS: Metabolic dysfunction-associated fatty liver disease (MAFLD) is associated with dyslipidemia and may promote cardiac lipotoxicity. Myocardial free fatty acids (FFA) oxidation (MOFFA) is normal in pre-diabetes, but reduced in heart failure. We hypothesized that during exercise MOFFA, very low-density lipoprotein triglycerides (VLDL-TG) secretion, hepatic FFA utilization, and lactate production differ among obese subjects with and without MAFLD. METHODS: Nine obese subjects with MAFLD and 8 matched subjects without MAFLD (Control) without a history of heart failure and cardiovascular disease were compared before and after 90-min exercise at 50% Peak oxygen consumption. Basal and exercise induced cardiac and hepatic FFA oxidation, uptake and re-esterification and VLDL-TG secretion were measured using [11C]palmitate positron-emission tomography and [1-14C]VLDL-TG. RESULTS: In the heart, increased MOFFA was observed after exercise in MAFLD, whereas MOFFA decreased in Control (basal vs exercise, MAFLD: 4.1 (0.8) vs 4.8 (0.8) µmol·100 ml-1 min-1; Control: 4.9 (1.8) vs 4.0 (1.1); µmol·100 ml-1 min-1, mean (SD), p < 0.048). Hepatic FFA fluxes were significantly lower in MAFLD than Control and increased ≈ two-fold in both groups. VLDL-TG secretion was 50% greater in MAFLD at rest and similarly suppressed during exercise. Plasma lactate increased significantly less in MAFLD than Control during exercise. CONCLUSIONS: Using robust tracer-techniques we found that obese subjects with MAFLD do not downregulate MOFFA during exercise compared to Control, possibly due to diminished lactate supply. Hepatic FFA fluxes are significantly lower in MAFLD than Control, but increase similarly with exercise. VLDL-TG export remains greater in MAFLD compared to Control. Basal and post-exercise myocardial and hepatic FFA, VLDL-TG and lactate metabolism is abnormal in subjects with MAFLD compared to Control.

Human translatability of the GAN diet-induced obese mouse model of non-alcoholic steatohepatitis.

BACKGROUND: Animal models of non-alcoholic steatohepatitis (NASH) are important tools in preclinical research and drug discovery. Gubra-Amylin NASH (GAN) diet-induced obese (DIO) mice represent a model of fibrosing NASH. The present study directly assessed the clinical translatability of the model by head-to-head comparison of liver biopsy histological and transcriptome changes in GAN DIO-NASH mouse and human NASH patients. METHODS: C57Bl/6 J mice were fed chow or the GAN diet rich in saturated fat (40%), fructose (22%) and cholesterol (2%) for ≥38 weeks. Metabolic parameters as well as plasma and liver biomarkers were assessed. Liver biopsy histology and transcriptome signatures were compared to samples from human lean individuals and patients diagnosed with NASH. RESULTS: Liver lesions in GAN DIO-NASH mice showed similar morphological characteristics compared to the NASH patient validation set, including macrosteatosis, lobular inflammation, hepatocyte ballooning degeneration and periportal/perisinusoidal fibrosis. Histomorphometric analysis indicated comparable increases in markers of hepatic lipid accumulation, inflammation and collagen deposition in GAN DIO-NASH mice and NASH patient samples. Liver biopsies from GAN DIO-NASH mice and NASH patients showed comparable dynamics in several gene expression pathways involved in NASH pathogenesis. Consistent with the clinical features of NASH, GAN DIO-NASH mice demonstrated key components of the metabolic syndrome, including obesity and impaired glucose tolerance. CONCLUSIONS: The GAN DIO-NASH mouse model demonstrates good clinical translatability with respect to the histopathological, transcriptional and metabolic aspects of the human disease, highlighting the suitability of the GAN DIO-NASH mouse model for identifying therapeutic targets and characterizing novel drug therapies for NASH.

Non-alcoholic fatty liver disease alters expression of genes governing hepatic nitrogen conversion.

BACKGROUND & AIMS: We recently showed that the functional capacity for ureagenesis is deficient in non-alcoholic fatty liver disease (NAFLD) patients. The aim of this study was to assess expression of urea cycle-related genes to elucidate a possible gene regulatory basis to the functional problem. METHODS: Liver mRNA expression analyses within the gene pathway governing hepatic nitrogen conversion were performed in 20 non-diabetic, biopsy-proven NAFLD patients (8 simple steatosis; 12 non-alcoholic steatohepatitis [NASH]) and 12 obese and 14 lean healthy individuals. Sixteen NAFLD patients were included for gene expression validation. Relationship between gene expressions and functional capacity for ureagenesis was described. RESULTS: Gene expression of most urea cycle-related enzymes were downregulated in NAFLD vs both control groups; markedly so for the urea cycle flux-generating carbamoyl phosphate synthetase (CPS1) (~3.5-fold, P < .0001). In NASH, CPS1 downregulation paralleled the deficit in ureagenesis (P = .03). Additionally, expression of several genes involved in amino acid uptake and degradation, and the glucagon receptor gene, were downregulated in NAFLD. Conversely, glutamine synthetase (GS) expression increased >1.5-fold (P ≤ .03), inversely related to CPS1 expression (P = .004). CONCLUSIONS: NAFLD downregulated the expression of urea cycle-related genes. Downregulation of urea cycle flux-generating CPS1 correlated with the loss of functional capacity for ureagenesis in NASH. On gene level, these changes coincided with an increase in the major ammonia scavenging enzyme GS. The effects seemed related to a fatty liver as such rather than NASH or obesity. The findings support gene regulatory mechanisms involved in the deficient ureagenesis of NAFLD, but it remains unexplained how hepatocyte fat accumulation exerts these effects.

Hepatic exposure of metformin in patients with non-alcoholic fatty liver disease.

AIMS: Metformin is first-line treatment of type 2 diabetes mellitus and reduces cardiovascular events in patients with insulin resistance and type 2 diabetes. Target tissue for metformin action is thought to be the liver, where metformin distribution depends on facilitated transport by polyspecific transmembrane organic cation transporters (OCTs). Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in the western world with strong associations to insulin resistance and the metabolic syndrome, but whether NAFLD affects metformin biodistribution to the liver is not known. In this study, the primary aim was to investigate in vivo hepatic uptake of metformin dynamically in humans with variable degrees of liver affection. As a secondary aim, we wished to correlate hepatic metformin distribution with OCT gene transcription determined in diagnostic liver biopsies. METHODS: Eighteen patients with biopsy-proven NAFLD were investigated using 11C-metformin PET/CT technique. Gene transcripts of OCTs were determined by real-time polymerase chain reaction (PCR). RESULTS: We observed similar hepatic volume of distribution of metformin between patients with simple steatosis and non-alcoholic steatohepatitis (NASH) (Vd 2.38 ± 0.56 vs. 2.10 ± 0.39, P = 0.3). There was no association between hepatic exposure to metformin and the degree of inflammation or fibrosis, and no clear correlation between metformin distribution and OCT gene transcription. CONCLUSION: Metformin is distributed to the liver in patients with NAFLD and the distribution is not impaired by inflammation or fibrosis. The findings imply that metformin action in liver in patients with NAFLD may be preserved.

[Treatment of non-alcoholic fatty liver disease].

Non-alcoholic fatty liver disease and especially non-alcoholic steatohepatitis (NASH) and fibrosis are associated with severe liver disease and increased cardiovascular risk. It is therefore important to identify patients with NASH fibrosis. Therapeutical options include life style intervention and pharmacological treatment with vitamin E and pioglitazone; however, evidence of effect is scarce for all options. New treatments are under investigation and include glucagon-like peptide-1, farnesoid receptor as well as peroxisome proliferator-activated receptor-α/δ agonists. Bariatric surgery may be an option in selected patients.

Niemann-Pick type C2 protein supplementation in experimental non-alcoholic fatty liver disease.

BACKGROUND AND AIMS: Hepatic cholesterol deposition drives inflammation and fibrosis in non-alcoholic steatohepatitis (NASH). The Niemann-Pick type C2 (NPC2) protein plays an important role in regulating intracellular cholesterol trafficking and homeostasis. We hypothesized that intravenous NPC2 supplementation reduces cholesterol accumulation, hepatic inflammation and fibrogenesis in a nutritional NASH rat model. METHODS: Rats were fed a high-fat, high-cholesterol (HFHC) diet for four weeks resulting in moderately severe NASH. Animals were treated with intravenous NPC2 or placebo twice weekly for either the last two weeks or the entire four weeks. End-points were liver/body- and spleen/body weight ratios, histopathological NASH scores, fibrosis, serum liver enzymes, cholesterol, lipoproteins, cytokines, and quantitative polymerase chain reaction derived hepatic gene expression related to cholesterol metabolism, inflammation, and fibrosis. RESULTS: HFHC rats developed hepatomegaly, non-fibrotic NASH histopathology, elevated liver enzymes, serum cholesterol, and pro-inflammatory cytokines. Their sterol regulatory element binding factor 2 (SREBF2) and low-density lipoprotein receptor (LDL-R) mRNAs were down-regulated compared with rats on standard chow. NPC2 did not improve liver weight, histopathology, levels of serum liver enzymes or pro-inflammatory tumor necrosis factor-α (TNFα), Interleukin (IL)-6, or IL-1ß in HFHC rats. Two weeks of NPC2 treatment lowered hepatic TNFα and COL1A1 mRNA expression. However, this effect was ultimately reversed following additional two weeks of treatment. Four weeks NPC2 treatment of rats raised ATP-binding cassette A1 (ABCA1) and low-density lipoprotein receptor (LDLR) mRNAs in the liver, concurrent with a strong tendency towards higher serum high-density lipoprotein (HDL). Furthermore, the peroxisome proliferator activated receptor-É£ (PPARG) gene expression was reduced. CONCLUSIONS: NPC2 proved inefficient at modifying robust hepatic NASH end-points in a HFHC NASH model. Nonetheless, our data suggest that hepatic ABCA1 expression and reverse cholesterol transport were upregulated by NPC2 treatment, thus presenting putative therapeutic effects in diseases associated with deregulated lipid metabolism.

Placebo-controlled, randomised clinical trial: high-dose resveratrol treatment for non-alcoholic fatty liver disease.

OBJECTIVE: "The obesity epidemic" has led to an increase in obesity-related conditions including non-alcoholic fatty liver disease (NAFLD), for which effective treatments are in demand. The polyphenol resveratrol prevents the development of experimental NAFLD through modulation of cellular pathways involved in calorie restriction. We aimed to test the hypothesis that resveratrol alleviates NAFLD in a randomised, clinical trial. MATERIALS AND METHODS: A total of 28 overweight patients with transaminasemia and histological NAFLD were randomised 1:1 to placebo or resveratrol 1.5 g daily for 6 months. Twenty-six participants completed the trial and underwent repeated clinical investigation, blood work, MR spectroscopy; and 19 participants agreed to a repeat liver biopsy. RESULTS: Resveratrol treatment was generally not superior to placebo in improving plasma markers of liver injury (primary outcome: alanine transaminase, p = 0.51). Resveratrol-treated patients showed a 3.8% decrease in liver lipid content (p = 0.03), with no difference between the two treatment arms (p = 0.38) and no improvement of histological features. Resveratrol treatment was not associated with improvements in insulin sensitivity or markers of the metabolic syndrome, except for a transient decrease in systolic BP. Microarray analysis and qRT-PCR revealed no major changes in expression profile. Also, we report a serious adverse event in a patient who developed fever and bicytopenia. CONCLUSIONS: In this placebo-controlled, high-dose and long-term study, resveratrol treatment had no consistent therapeutic effect in alleviating clinical or histological NAFLD, though there may be a small ameliorating effect on liver function tests and liver fat accumulation.

Plasma levels of OLFM4 in normals and patients with gastrointestinal cancer.

Olfactomedin 4 (OLFM4) is a secreted glycoprotein predominantly expressed in bone marrow and gastrointestinal tissues. Aberrant expression of OLFM4 has been shown in several cancers. However, the clinical significance hereof is currently controversial. OLFM4 has been proposed as a candidate biomarker of gastrointestinal cancers. To address this, we developed monoclonal antibodies against synthetic peptides representing various segments of OLFM4. We examined expression of OLFM4 in epithelial cells by immunohistochemistry and found that OLFM4 is highly expressed in proliferating benign epithelial cells and in some carcinoma cells. We developed an Enzyme Linked Immunosorbent Assay for OLFM4 and investigated whether plasma levels of OLFM4 reflect colorectal malignancies, but were unable to see any such association. Instead, we observed two populations of individuals with respect to OLFM4 levels in plasma, the majority with OLFM4 in plasma between 0 and 0.1 µg/ml, mean 0.028 µg/ml while 10% of both normals and patients with cancers had OLFM4 between 4 and 60 µg/ml, mean 15 µg/ml. The levels were constant over time. The background for this high plasma level is not known, but must be taken into account if OLFM4 is used as biomarker for GI cancers.

Effect of resveratrol on experimental non-alcoholic steatohepatitis.

Non-alcoholic fatty liver disease and non-alcoholic steatohepatitis (NASH) are increasing clinical problems for which effective treatments are required. The polyphenol resveratrol prevents the development of fatty liver disease in a number of experimental studies. We hypothesized that it could revert steatohepatitis, including hepatic inflammation and fibrosis, in an experimental NASH model. To induce hepatic steatohepatitis, a 65% fat, 2% cholesterol and 0.5% cholate (HFC) diet was fed to rats for 1 or 16 weeks, prior to treatment. Subsequently, the diet was supplemented with resveratrol (approx. 100mg/rat/day) to three intervention groups; week 2-4, 2-7 or 17-22. Treated animals were sacrificed at the end of each intervention period with appropriate control and HFC diet controls. Blood and liver were harvested for analysis. When commenced early, resveratrol treatment partially mitigated transaminase elevations, hepatic enlargement and TNFα induced protein-3 protein expression, but generally resveratrol treatment had no effect on elevated hepatic triglyceride levels, histological steatohepatitis or fibrosis. We observed a slight reduction in Collagen1α1 mRNA expression and no reduction in the mRNA expression of other markers of fibrosis, inflammation or steatosis (TGFß, TNFα, α2-MG, or SREBP-1c). Resveratrol metabolites were detected in serum, including trans-resveratrol-3-O-sulphate/trans-resveratrol-4'-O-sulphate (mean concentration 7.9 µg/ml). Contrary to the findings in experimental steatosis, resveratrol treatment had no consistent therapeutic effect in alleviating manifest experimental steatohepatitis.

Effects of resveratrol in experimental and clinical non-alcoholic fatty liver disease.

The prevalence of obesity and related conditions like non-alcoholic fatty liver disease (NAFLD) is increasing worldwide and therapeutic options are limited. Alternative treatment options are therefore intensively sought after. An interesting candidate is the natural polyphenol resveratrol (RSV) that activates adenosinmonophosphate-activated protein kinase (AMPK) and silent information regulation-2 homolog 1 (SIRT1). In addition, RSV has known anti-oxidant and anti-inflammatory effects. Here, we review the current evidence for RSV-mediated effects on NAFLD and address the different aspects of NAFLD and non-alcoholic steatohepatitis (NASH) pathogenesis with respect to free fatty acid (FFA) flux from adipose tissue, hepatic de novo lipogenesis, inadequate FFA ß-oxidation and additional intra- and extrahepatic inflammatory and oxidant hits. We review the in vivo evidence from animal studies and clinical trials. The abundance of animal studies reports a decrease in hepatic triglyceride accumulation, liver weight and a general improvement in histological fatty liver changes, along with a reduction in circulating insulin, glucose and lipid levels. Some studies document AMPK or SIRT1 activation, and modulation of relevant markers of hepatic lipogenesis, inflammation and oxidation status. However, AMPK/SIRT1-independent actions are also likely. Clinical trials are scarce and have primarily been performed with a focus on overweight/obese participants without a focus on NAFLD/NASH and histological liver changes. Future clinical studies with appropriate design are needed to clarify the true impact of RSV treatment in NAFLD/NASH patients.

[Prognosis and treatment of non-alcoholic fatty liver disease and steatohepatitis]. / Prognose og behandling af nonalkoholisk fedtleversygdom og steatohepatitis.

Non-alcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH) are among the most common liver diseases. NAFLD with pure steatosis is considered benign. NASH, however, may progress to liver cirrhosis with risk of liver cancer. Therefore, it is important to search for treatment modalities for NASH. Because obesity and insulin resistance are risk factors for NASH, weight loss and exercise are considered mandatory, although limited data are in support. Presently, we focus on the prognosis of NAFLD and NASH and on pharmaceutical treatment, especially in the form of vitamin E and pioglitazone.

Smarcc1 expression: a significant predictor of disease-specific survival in patients with clinically localized prostate cancer treated with no intention to cure.

OBJECTIVE: The clinical outcome of prostate cancer (PC) is extremely variable and therefore difficult to predict at the early stage of the disease. Since curative-intended therapies are bound up with the risk of severe adverse events, identification of new prognostic markers in PC is essential in individualized clinical treatment. The Smarcc1 protein, a part of the intranuclear SWI/SNF complex, is up-regulated in PC, and has been suggested to be implicated in tumour dedifferentiation, progression and biochemical recurrence. This makes Smarcc1 a possible candidate marker for PC survival. MATERIAL AND METHODS: Immunohistochemistry was used to measure protein expression levels of Smarcc1in on a tissue microarray containing specimens from 100 patients suffering from clinically localized PC treated with no intention to cure and followed to death. RESULTS: The median age at diagnosis was 75.5 years (55-95 years) and the median survival time was 5 years (0.01-15 years). In total, 41 patients (41%) died of PC. Statistically, there was no significant association between Smarcc1 immunostaining (negative/positive) and Gleason score (p = 0.7/0.8) or the clinical T stage (p = 0.9). Positive staining for Smarcc1 in patients with clinically localized PC correlated with a prolonged disease-free survival as opposed to negative staining (p = 0.025). CONCLUSION: In patients with clinically localized PC treated without intention of cure, Smarcc1 expression was a statistically significant and independent predictor of disease-specific survival.

Genetic and epigenetic SLC18A2 silencing in prostate cancer is an independent adverse predictor of biochemical recurrence after radical prostatectomy.

PURPOSE: This study investigates SLC18A2 (vesicular monoamine transporter 2) expression in prostate adenocarcinoma and examines its potential as a predictive marker for prostate cancer patient outcome after radical prostatectomy. EXPERIMENTAL DESIGN: Expression and single nucleotide polymorphism microarray analyses identified SLC18A2 as both down-regulated and subject to common loss-of-heterozygosity in prostate cancer. Down-regulated SLC18A2 expression was validated on tissue microarrays containing benign and malignant prostate specimens from an independent patient group (n=738). Furthermore, SLC18A2 immunoreactivity in radical prostatectomy tumor specimens (n=506) was correlated to clinicopathologic characteristics and recurrence-free survival. The possibility of SLC18A2 silencing by aberrant DNA methylation in prostate cancer cells was investigated by bisulfite sequencing. RESULTS: Tissue microarray analysis revealed markedly lower cytoplasmic SLC18A2 staining in cancer compared with nonmalignant prostate tissue samples, confirming RNA expression profiling results. Furthermore, multivariate analysis identified cytoplasmic SLC18A2 immunoreactivity as a novel predictor of biochemical recurrence following prostatectomy (hazard ratio, 0.485; 95% confidence interval, 0.333-0.709; P<0.001) independent of prostate-specific antigen, Gleason score, tumor stage, and surgical margin status. SLC18A2 showed loss-of-heterozygosity in 23% of the tumors and was densely hypermethylated in 15 of 17 (88%) prostate cancer samples plus 6 of 6 prostate cancer cell lines. In contrast, SLC18A2 was unmethylated in 4 of 4 adjacent nonmalignant prostate and 3 of 5 benign prostatic hyperplasia tissue samples, whereas 2 of 5 benign prostatic hyperplasia samples had monoallelic hypermethylation. Methylation and histone deacetylase inhibitory agents rescued SLC18A2 expression in three prostate cancer cell lines. CONCLUSIONS: SLC18A2 silencing by DNA hypermethylation and/or allelic loss is a frequent event in prostate cancer and a novel independent predictor of biochemical recurrence after prostatectomy.

Snail1 is over-expressed in prostate cancer.

Transcription factor Snail1 is a mediator of cell migration and survival, and expression is elevated in several cancer types. The Snail1 gene is reportedly amplified in prostate cancer (PC), and we investigated Snail1 expression in PC. Immunohistochemical Snail1 staining was determined on a tissue microarray which includes 327 specimens of PC, 30 specimens from patients with benign prostatic hyperplasia (BPH), benign tissue from 30 PC patients and 15 high-grade prostate intraepithelial neoplasia (high-grade PIN) specimens. Clinicopathological and follow-up data were available for all patients. No BPH specimen and only 21% of benign tissue from PC patients showed high expression of Snail1. Only 7% of high-grade PIN patients expressed a high level of Snail1. In contrast, approximately 50% of PC tissue from patients with PC showed marked nuclear immunostaining. Snail1 immunostaining was significantly associated with Gleason score (p<0.05). Snail1 expression was not correlated to T stage, metastasis at time of diagnosis, risk of or time to recurrence. Snail1 expression was significantly increased in PC with a positive correlation to dedifferentiation, but not to cancer progression or prognosis. The presented data indicate that Snail1 expression is upregulated from the early stages of PC.

Intrinsic markers of tumour hypoxia and angiogenesis in localised prostate cancer and outcome of radical treatment: a retrospective analysis of two randomised radiotherapy trials and one surgical cohort study.

BACKGROUND: Expression of intrinsic markers of tumour hypoxia and angiogenesis are important predictors of radiotherapeutic, and possibly surgical, outcome in several cancers. Extent of tumour hypoxia in localised prostate cancer is comparable to that in other cancers, but few data exist on the association of extent of tumour hypoxia with treatment outcome. We aimed to study the predictive value of intrinsic markers of tumour hypoxia and angiogenesis in localised prostate cancer, both in patients treated with radiotherapy and in those treated surgically. METHODS: We applied a new, needle biopsy tissue microarray (TMA) technique to study diagnostic samples from men with localised, previously untreated prostate cancer treated in two randomised controlled trials of radiotherapy-dose escalation. Multivariate analysis by Cox proportional hazards was done to assess the association between clinical outcome, in terms of biochemical control, and immunohistochemical staining of hypoxia inducible factor-1 alpha (HIF-1 alpha), vascular endothelial growth factor (VEGF), and osteopontin expression. The analysis was repeated on an independent series of men with localised, previously untreated prostate cancer treated by radical prostatectomy. The main outcome was time to biochemical (ie, prostate-specific antigen [PSA]) failure. FINDINGS: Between Oct 12, 1995, and Feb 5, 2002, 308 patients were identified from two prospective, randomised trials at the Royal Marsden Hospital, London and Sutton, UK, for the radiotherapy cohort and diagnostic biopsies were available for 201 of these patients. Between June 6, 1995, and Nov 4, 2005, 329 patients were identified from the Aarhus University Hospital, Skejby, Denmark, for the prostatectomy cohort; of these, 40 patients were excluded because the tumour was too small to sample (19 patients), because the paraffin block was too thin (19 patients), or because the blocks were missing (two patients), leaving 289 patients for analysis. For patients treated with radiotherapy, increased staining for VEGF (p=0.008) and HIF-1 alpha (p=0.02) expression, but not increased osteopontin expression (p=0.978), were significant predictors of a shorter time to biochemical failure on multivariate analysis, independent of clinical tumour stage, Gleason score, serum PSA concentration, and dose of radiotherapy. For patients treated with surgery, increased staining for VEGF (p<0.0001) and HIF-1 alpha (p<0.0001) expression, and increased osteopontin expression (p=0.0005) were each significantly associated with a shorter time to biochemical failure on multivariate analysis, independent of pathological tumour stage, Gleason score, serum PSA concentration, and margin status. INTERPRETATION: To our knowledge, this is the largest study of intrinsic markers of hypoxia and angiogenesis in relation to the outcome of radical treatment of localised prostate cancer. Increased expression of VEGF, HIF-1 alpha, and, for patients treated with surgery, osteopontin, identifies patients at high risk of biochemical failure who would be suitable for enrolment into trials of treatment intensification.

Prostate cancer: to scan or not to scan for lymph node involvement?

OBJECTIVE: When diagnosing lymph node involvement in prostate cancer (PC), the available imaging techniques are considered to be of doubtful value but are still in common use in many institutions because of a lack of better alternatives. To help us choose between methods while we wait for improvements in technologies, we evaluated the performance of ultrasonography (US), CT and MRI. MATERIAL AND METHODS: We reviewed the records of all PC patients who were potential candidates for a surgical staging procedure prior to treatment with intent to cure at Skejby Sygehus, Denmark between 1996 and 2004. In total, 227 patients met our inclusion criteria and 149 were examined with US, 80 with MRI and 71 with CT. The performance of the three modalities was analysed. RESULTS: A total of 50 patients (22%) had lymph node metastases (pN1) and only 12 of these cases (24%) were detected by imaging. US identified all 12 pN1 patients, MRI five and CT did not identify any. Moreover, US missed 28 patients with pN1 metastases, MRI 18 and CT 13. Consequently, US, MRI and CT had sensitivities of 0.3, 0.22 and 0, respectively, while all three modalities had a specificity of 1. The 12 patients identified by imaging had clinically advanced and pathologically aggressive cancers. CONCLUSIONS: Available imaging techniques showed moderate to very low sensitivity. US proved superior to MRI and CT, but should only be applied in high-risk patients. In particular, CT can be considered unsuitable for diagnosing pelvic lymph node metastases in PC.