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Purpose: Recent advances in machine learning have enabled better understanding of large and complex visual data. Here, we aim to investigate patient outcome prediction with a machine learning method using only an image of tumour sample as an input., Methods: Utilising tissue microarray (TMA) samples obtained from the primary tumour of patients (N = 1299) within a nationwide breast cancer series with long-term-follow-up, we train and validate a machine learning method for patient outcome prediction. The prediction is performed by classifying samples into low or high digital risk score (DRS) groups. The outcome classifier is trained using sample images of 868 patients and evaluated and compared with human expert classification in a test set of 431 patients., Results: In univariate survival analysis, the DRS classification resulted in a hazard ratio of 2.10 (95% CI 1.33-3.32, p = 0.001) for breast cancer-specific survival. The DRS classification remained as an independent predictor of breast cancer-specific survival in a multivariate Cox model with a hazard ratio of 2.04 (95% CI 1.20-3.44, p = 0.007). The accuracy (C-index) of the DRS grouping was 0.60 (95% CI 0.55-0.65), as compared to 0.58 (95% CI 0.53-0.63) for human expert predictions based on the same TMA samples., Conclusions: Our findings demonstrate the feasibility of learning prognostic signals in tumour tissue images without domain knowledge. Although further validation is needed, our study suggests that machine learning algorithms can extract prognostically relevant information from tumour histology complementing the currently used prognostic factors in breast cancer.

Background: Detection of lymph node metastases is essential in breast cancer diagnostics and staging, affecting treatment and prognosis. Intraoperative microscopy analysis of sentinel lymph node frozen sections is standard for detection of axillary metastases but requires access to a pathologist for sample analysis. Remote analysis of digitized samples is an alternative solution but is limited by the requirement for high-end slide scanning equipment., Objective: To determine whether the image quality achievable with a low-cost, miniature digital microscope scanner is sufficient for detection of metastases in breast cancer lymph node frozen sections., Methods: Lymph node frozen sections from 79 breast cancer patients were digitized using a prototype miniature microscope scanner and a high-end slide scanner. Images were independently reviewed by two pathologists and results compared between devices with conventional light microscopy analysis as ground truth., Results: Detection of metastases in the images acquired with the miniature scanner yielded an overall sensitivity of 91% and specificity of 99% and showed strong agreement when compared to light microscopy (k = 0.91). Strong agreement was also observed when results were compared to results from the high-end slide scanner (k = 0.94). A majority of discrepant cases were micrometastases and sections of which no anticytokeratin staining was available., Conclusion: Accuracy of detection of metastatic cells in breast cancer sentinel lymph node frozen sections by visual analysis of samples digitized using low-cost, point-of-care microscopy is comparable to analysis of digital samples scanned using a high-end, whole slide scanner. This technique could potentially provide a workflow for digital diagnostics in resource-limited settings, facilitate sample analysis at the point-of-care and reduce the need for trained experts on-site during surgical procedures., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: Johan Lundin and Mikael Lundin are founders and co-owners of Fimmic Oy, Helsinki, Finland. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Background: Previously, we have been able to demonstrate the possibility of coating the inner surface of the renal arteries in porcine kidneys with a heparin conjugate during hypothermic machine perfusion (HMP). The purpose of this study was to assess the efficacy of this treatment in reducing early ischemia-reperfusion injury., Method: Brain death was induced in male landrace pigs by stepwise volume expansion of an epidural balloon catheter until negative cerebral perfusion pressure (CPP) was obtained. Both kidneys (matched pairs; n = 6 + 6) were preserved for 20 hours by HMP during which 50 mg heparin conjugate was added to one of the HMP systems (treated group). A customized ex vivo normothermic oxygenated perfusion (NP) system with added exogenous creatinine was used to evaluate early kidney function. Blood, urine and histological samples were collected during the subsequent 3 hours of NP., Results: Kidney weight was lower at the end of NP (P = 0.017) in the treated group compared with control kidneys. The rate of decline in creatinine level was faster (P = 0.024), total urinary volume was higher (P = 0.031), and the level of urine neutrophil gelatinase-associated lipocalin (NGAL) was lower (P = 0.031) in the treated group. Histologically, less tubular changes were seen (P = 0.046). During NP intrarenal resistance remained lower (P < 0.0001) in the treated group., Conclusions: Perfusion of porcine kidneys with heparin conjugate during HMP reduces preservation injury and improves organ function shortly after reperfusion. No increased risk of bleeding was seen in this setup. This protective strategy may potentially improve the quality of transplanted kidneys in the clinical setting.

BACKGROUND Improved understanding of the impact of kidney transplantation on healthcare resource use/costs and loss of productivity could aid decision making about funding allocation and resources needed for the treatment of chronic kidney disease in stage 5. MATERIAL AND METHODS This was a retrospective study utilizing data from Swedish national health registers of patients undergoing kidney transplantation. Primary outcomes were renal disease-related healthcare resource utilization and costs during the 5 years after transplantation. Secondary outcomes included total costs and loss of productivity. Regression analysis identified factors that influenced resource use, costs, and loss of productivity. RESULTS During the first year after transplantation, patients (N=3120) spent a mean of 25.7 days in hospital and made 21.6 outpatient visits; mean renal disease-related total cost was €66,014. During the next 4 years, resource use was approximately 70% (outpatient) to 80% (inpatient) lower, and costs were 75% lower. Before transplantation, 62.8% were on long-term sick leave, compared with 47.4% 2 years later. Higher resource use and costs were associated with age <10 years, female sex, graft from a deceased donor, prior hemodialysis, receipt of a previous transplant, and presence of comorbidities. Higher levels of sick leave were associated with female sex, history of hemodialysis, and type 1 diabetes. Overall 5-year graft survival was 86.7% (95% CI 85.3-88.2%). CONCLUSIONS After the first year following transplantation, resource use and related costs decreased, remaining stable for the next 4 years. Demographic and clinical factors, including age <10 years, female sex, and type 1 diabetes were associated with higher costs and resource use.

Background: Radical prostatectomy reduces mortality among men with clinically detected localized prostate cancer, but evidence from randomized trials with long-term follow-up is sparse., Methods: We randomly assigned 695 men with localized prostate cancer to watchful waiting or radical prostatectomy from October 1989 through February 1999 and collected follow-up data through 2017. Cumulative incidence and relative risks with 95% confidence intervals for death from any cause, death from prostate cancer, and metastasis were estimated in intention-to-treat and per-protocol analyses, and numbers of years of life gained were estimated. We evaluated the prognostic value of histopathological measures with a Cox proportional-hazards model., Results: By December 31, 2017, a total of 261 of the 347 men in the radical-prostatectomy group and 292 of the 348 men in the watchful-waiting group had died; 71 deaths in the radical-prostatectomy group and 110 in the watchful-waiting group were due to prostate cancer (relative risk, 0.55; 95% confidence interval [CI], 0.41 to 0.74; P<0.001; absolute difference in risk, 11.7 percentage points; 95% CI, 5.2 to 18.2). The number needed to treat to avert one death from any cause was 8.4. At 23 years, a mean of 2.9 extra years of life were gained with radical prostatectomy. Among the men who underwent radical prostatectomy, extracapsular extension was associated with a risk of death from prostate cancer that was 5 times as high as that among men without extracapsular extension, and a Gleason score higher than 7 was associated with a risk that was 10 times as high as that with a score of 6 or lower (scores range from 2 to 10, with higher scores indicating more aggressive cancer)., Conclusions: Men with clinically detected, localized prostate cancer and a long life expectancy benefited from radical prostatectomy, with a mean of 2.9 years of life gained. A high Gleason score and the presence of extracapsular extension in the radical prostatectomy specimens were highly predictive of death from prostate cancer. (Funded by the Swedish Cancer Society and others.).

Prostate cancer is a highly heterogeneous disease and the clinical outcome is varying. While current prognostic tools are regarded insufficient, there is a critical need for markers that would aid prognostication and patient risk-stratification. Heat shock transcription factor 1 (HSF1) is crucial for cellular homeostasis, but also a driver of oncogenesis. The clinical relevance of HSF1 in prostate cancer is, however, unknown. Here, we identified HSF1 as a potential biomarker in mRNA expression datasets on prostate cancer. Clinical validation was performed on tissue microarrays from independent cohorts: one constructed from radical prostatectomies from 478 patients with long term follow-up, and another comprising of regionally advanced to distant metastatic samples. Associations with clinical variables and disease outcomes were investigated. Increased nuclear HSF1 expression correlated with disease advancement and aggressiveness and was, independently from established clinicopathological variables, predictive of both early initiation of secondary therapy and poor disease-specific survival. In a joint model with the clinical Cancer of the Prostate Risk Assessment post-Surgical (CAPRA-S) score, nuclear HSF1 remained a predictive factor of shortened disease-specific survival. The results suggest that nuclear HSF1 expression could serve as a novel prognostic marker for patient risk-stratification on disease progression and survival after radical prostatectomy., Competing Interests: CONFLICTS OF INTEREST The authors declare that they have no conflicts of interest.

Ischemia reperfusion injury is one of the major complications responsible for delayed graft function in kidney transplantation. Applications to reduce reperfusion injury are essential due to the widespread use of kidneys from deceased organ donors where the risk for delayed graft function is especially prominent. We have recently shown that coating of inflamed or damaged endothelial cells with a unique heparin conjugate reduces thrombosis and leukocyte recruitment. In this study we evaluated the binding capacity of the heparin conjugate to cultured human endothelial cells, to kidneys from brain-dead porcine donors, and to murine kidneys during static cold storage. The heparin conjugate was able to stably bind cultured endothelial cells with high avidity, and to the renal vasculature of explanted kidneys from pigs and mice. Treatment of murine kidneys prior to transplantation reduced platelet deposition and leukocyte infiltration 24 hours post-transplantation, and significantly improved graft function. The present study thus shows the benefits of enhanced protection of the renal vasculature during cold storage, whereby increasing the antithrombotic and anti-adhesive properties of the vascular endothelium yields improved renal function early after transplantation.

Image-based machine learning and deep learning in particular has recently shown expert-level accuracy in medical image classification. In this study, we combine convolutional and recurrent architectures to train a deep network to predict colorectal cancer outcome based on images of tumour tissue samples. The novelty of our approach is that we directly predict patient outcome, without any intermediate tissue classification. We evaluate a set of digitized haematoxylin-eosin-stained tumour tissue microarray (TMA) samples from 420 colorectal cancer patients with clinicopathological and outcome data available. The results show that deep learning-based outcome prediction with only small tissue areas as input outperforms (hazard ratio 2.3; CI 95% 1.79-3.03; AUC 0.69) visual histological assessment performed by human experts on both TMA spot (HR 1.67; CI 95% 1.28-2.19; AUC 0.58) and whole-slide level (HR 1.65; CI 95% 1.30-2.15; AUC 0.57) in the stratification into low- and high-risk patients. Our results suggest that state-of-the-art deep learning techniques can extract more prognostic information from the tissue morphology of colorectal cancer than an experienced human observer.

Caveolin-1 (CAV1) is over-expressed in prostate cancer (PCa) and is associated with adverse prognosis, but the molecular mechanisms linking CAV1 expression to disease progression are poorly understood. Extensive gene expression correlation analysis, quantitative multiplex imaging of clinical samples, and analysis of the CAV1-dependent transcriptome, supported that CAV1 re-programmes TGFβ signalling from tumour suppressive to oncogenic (i.e. induction of SLUG, PAI-1 and suppression of CDH1, DSP, CDKN1A). Supporting such a role, CAV1 knockdown led to growth arrest and inhibition of cell invasion in prostate cancer cell lines. Rationalized RNAi screening and high-content microscopy in search for CAV1 upstream regulators revealed integrin beta1 (ITGB1) and integrin associated proteins as CAV1 regulators. Our work suggests TGFβ signalling and beta1 integrins as potential therapeutic targets in PCa over-expressing CAV1, and contributes to better understand the paradoxical dual role of TGFβ in tumour biology.

Background: The spread of vector-borne diseases to new regions has become a global threat due to climate change, increasing traffic, and movement of people and animals. Dirofilaria repens, the canine subcutaneous filarioid nematode, has expanded its distribution range northward during the last decades. The northernmost European locations, where the parasite life-cycle has been confirmed, are Estonia and the Novgorod Region in Russia., Results: Herein, we describe an autochthonous D. repens infection in a Finnish woman. We also present two cases of D. repens infection in imported dogs indicating the life-cycle in the Russian Vyborg and St Petersburg areas, close to the Finnish border., Conclusions: The most obvious limiting factor of the northern distribution of D. repens is the summer temperature, due to the temperature-dependent development of larvae in vectors. With continuing climate change, further spread of D. repens in Fennoscandia can be expected.

Corline Heparin Conjugate (CHC), a compound of multiple unfractionated heparin chains, coats cells with a glycocalyx-like layer and may inhibit (xeno)transplant-associated activation of the plasma cascade systems. Here, we investigated the use of CHC to protect WT and genetically modified (GTKO.hCD46.hTBM) pig aortic endothelial cells (PAEC) in two pig-to-human in vitro xenotransplantation settings. Model 1: incubation of untreated or hTNFα-treated PAEC with 10% human plasma induced complement C3b/c and C5b-9 deposition, cellular activation and coagulation activation in WT and GTKO.hCD46.hTBM PAEC. Coating of untreated or hTNFα-treated PAEC with CHC (100 µg/ml) protected against human plasma-induced endothelial activation and damage. Model 2: PAEC were grown on microcarrier beads, coated with CHC, and incubated with non-anticoagulated whole human blood. Genetically modified PAEC significantly prolonged clotting time of human blood (115.0 ± 16.1 min, p < 0.001) compared to WT PAEC (34.0 ± 8.2 min). Surface CHC significantly improved the human blood compatibility of PAEC, as shown by increased clotting time (WT: 84.3 ± 11.3 min, p < 0.001; GTKO.hCD46.hTBM: 146.2 ± 20.4 min, p < 0.05) and reduced platelet adhesion, complement activation, coagulation activation and inhibition of fibrinolysis. The combination of CHC coating and genetic modification provided the greatest compatibility with human blood, suggesting that pre-transplant perfusion of genetically modified porcine organs with CHC may benefit post-transplant xenograft function.

Objectives: Since the early 1990s, low long-term survival rates following pancreatic surgery for pancreatic ductal adenocarcinoma have challenged us to improve treatment. In this series, we aim to show improved survival from pancreatic ductal adenocarcinoma during the era of centralized pancreatic surgery., Methods: Analysis of all pancreatic resections performed at Helsinki University Hospital and survival of pancreatic ductal adenocarcinoma patients during 2000-2013 were included. Post-operative complications such as fistulas, reoperations, and mortality rates were recorded. Patient and tumor characteristics were compared with survival data., Results: Of the 853 patients undergoing pancreatic surgery, 581 (68%) were pancreaticoduodenectomies, 195 (21%) distal resections, 28 (3%) total pancreatectomies, and 49 (6%) other procedures. Mortality after pancreaticoduodenectomy was 2.1%. The clinically relevant B/C fistula rate was 7% after pancreaticoduodenectomy and 13% after distal resection, and the re-operation rate was 5%. The 5- and 10-year survival rates for pancreatic ductal adenocarcinoma were 22% and 14%; for T1-2, N0 and R0 tumors, the corresponding survival rates were 49% and 31%. Carbohydrate antigen 19-9 >75 kU/L, carcinoembryonic antigen >5 µg/L, N1, lymph-node ratio >20%, R1, and lack of adjuvant therapy were independent risk factors for decreased survival., Conclusion: After centralization of pancreatic surgery in southern Finland, we have managed to enable pancreatic ductal adenocarcinoma patients to survive markedly longer than in the early 1990s. Based on a 1.7-million population in our clinic, mortality rates are equal to those of other high-volume centers and long-term survival rates for pancreatic ductal adenocarcinoma have now risen to some of the highest reported.

The clinical course of prostate cancer is highly variable. Current prognostic variables, stage, and Gleason score have limitations in assessing treatment regimens for individual patients, especially in the intermediate-risk group of Gleason score 7. ERG:TMPRSS2 fusion and loss of PTEN are some of the most common genetic alterations in prostate cancer. Immunohistochemistry of PTEN and ERG has generated interest as a promising method for more precise outcome prediction but requires further validation in population-based cohorts. We studied the predictive value of ERG and PTEN expression by immunohistochemistry in two large radical prostatectomy cohorts comprising 815 patients with extensive follow-up information. Clinical end points were initiation of secondary therapy, overall survival, and disease-specific survival. Predictions of clinical outcomes were also assessed according to androgen receptor (AR) activity. PTEN loss, especially in ERG-negative cancers, predicted initiation of secondary treatments and shortened disease-specific survival time, as well as stratifying Gleason score 7 patients into different prognostic groups with regard to secondary treatments and disease-specific survival. High AR immunoreactivity in ERG-negative cancers with PTEN loss predicted worse disease-specific survival. We also observed that in Gleason score 7 ERG-negative cases with PTEN loss and high AR expression have significantly shorter disease-specific survival time compared with ERG-positive cases. Our conclusion is that loss of PTEN is a strong determining factor for shorter disease-specific survival time and initiation of secondary therapies after radical prostatectomy. The predictive value of PTEN immunoreactivity is further accentuated in ERG-negative cancers with high AR expression. Negative PTEN expression, accompanied by ERG status, can be used to stratify patients with Gleason score 7 into different survival groups. Assessment of PTEN and ERG status could provide an additional tool for initial diagnostics when determining the prognosis and subsequent follow-up regimen for patients treated by radical prostatectomy.

Background: The Wnt/β-catenin pathway has a key role in regulating cellular processes and its aberrant signaling can lead to cancer development. The role of β-catenin expression in pancreatic ductal adenocarcinoma is somewhat controversial. Transcription factor PROX1 is a target of Wnt/β-catenin signaling and it is involved in carcinogenesis through alterations in its expression. The actions can be either oncogenic or tumor suppressive depending on the tissue. The aim of this study was to investigate PROX1 and β-catenin expression in pancreatic ductal adenocarcinoma (PDAC)., Methods: Expression of PROX1 and β-catenin were evaluated in 156 patients by immunohistochemistry of tissue microarrays. Associations between tumor marker expression and clinicopathological parameters were assessed by the Fischer's exact-test or the linear-by-linear association test. The Kaplan-Meier method and log-rank test were used for survival analysis. Uni- and multivariate survival analyses were carried out by the Cox regression proportional hazard model., Results: High PROX1 expression was seen in 74 (48 %) tumors, and high β-catenin expression in 100 (65 %). High β-catenin expression was associated with lower tumor grade (p = 0.025). High PROX1 and β-catenin expression associated significantly with lower risk of death from PDAC in multivariate analysis (HR = 0.63; 95 % CI 0.42-0.95, p = 0.026; and HR = 0.54; 95 % CI 0.35-0.82, p = 0.004; respectively). The combined high expression of PROX1 and β-catenin also predicted lower risk of death from PDAC (HR = 0.46; 95 % CI 0.28-0.76, p = 0.002)., Conclusion: In conclusion, high PROX1 and β-catenin expression were independent factors for better prognosis in pancreatic ductal adenocarcinoma.

The impact of complement activation and its possible relation to cytokine responses during malaria pathology was investigated in plasma samples from patients with confirmed Plasmodium falciparum malaria and in human whole-blood specimens stimulated with malaria-relevant agents ex vivo. Complement was significantly activated in the malaria cohort, compared with healthy controls, and was positively correlated with disease severity and with certain cytokines, in particular interleukin 8 (IL-8)/CXCL8. This was confirmed in ex vivo-stimulated blood specimens, in which complement inhibition significantly reduced IL-8/CXCL8 release. P. falciparum malaria is associated with systemic complement activation and complement-dependent release of inflammatory cytokines, of which IL-8/CXCL8 is particularly prominent., (© The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Background: Distinguishing between pancreatic cancer and chronic pancreatitis (CP) is often difficult. Certain (5-6%) CP cases are autoimmune in nature, and these patients respond to corticosteroid treatment, making surgery avoidable. Our aim was to evaluate the incidence of autoimmune pancreatitis (AIP) among patients operated on for a pancreatic mass with a final histology of CP., Patients and Methods: A total of 33 patients were operated on at the Tampere or Helsinki University Hospital for suspicion of cancer, but with final histopathological diagnosis of CP. The median age was 58 (31-81) years; 26 patients (79%) were male. There were 28 pancreaticoduodenectomes and five left pancreatic resections. Surgical specimens were re-evaluated by experienced pathologists, with representative samples chosen for immunohistochemistry Each sample was scored as positive or negative for immunoglobulin G4 (IgG4) independently by two pathologists. Honolulu consensus criteria served for AIP sub-typing., Results: Out of the 33 specimens, 10 (30%) were positive for IgG4. Histopathological re-evaluation of these revealed all to be type 1 AIP., Conclusion: The proportion of AIP, according to IgG4-positive immunohistochemistry and histological re-evaluation, was much higher than expected. This suggests that by focusing on diagnosis of AIP preoperatively, certain patients might be treated with corticosteroids and possibly avoid unnecessary surgery., (Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Aim of the Study: Podocalyxin-like 1 is a transmembrane glyco-protein whose overexpression associates in many cancers with poor prognosis and unfavorable clinicopathological characteristics. Until now, its prognostic value has never been studied in pancreatic ductal adenocarcinoma (PDAC). The aim of this study was to investigate podocalyxin expression in PDAC by a novel monoclonal antibody and a commercially available polyclonal antibody., Patients and Materials: With tissue microarrays and immuno-histochemistry, podocalyxin expression evaluation involved 168 PDAC patients. The associations of the podocalyxin tumor expression with clinicopathological variables were explored by Fisher's exact test and the linear-by-linear test. Survival analyses were by Kaplan-Meier analysis and the Cox proportional hazard model., Results: The polyclonal antibody revealed membranous podocalyxin expression in 73 (44.0%) specimens and the monoclonal antibody was highly expressed in 36 (21.8%) cases. Membranous expression by the polyclonal antibody was associated with T classification (p=0.045) and perineural invasion (p=0.005), and high expression by the mono-clonal antibody with poor differentiation (p=0.033). High podocalyxin expression associated significantly with higher risk of death from PDAC by both the polyclonal antibody (hazard ratio (HR) = 1.62; 95% confidence interval (CI) 1.12-2.33; p=0.01) and the monoclonal antibody (HR = 2.10, 95% CI 1.38-3.20; p<0.001). The results remained significant in multivariate analysis, adjusted for age, gender, stage, lymph node ratio (≥/< 20%), and perivascular invasion (respectively as HR = 2.03; 95% CI 1.32-3.13, p=0.001; and as HR = 2.36; 95% CI 1.47-3.80, p<0.001)., Conclusion: We found podocalyxin to be an independent factor for poor prognosis in PDAC. To our knowledge, this is the first such report of its prognostic value.

Ischaemia-reperfusion injury (IRI) poses a major challenge in many thrombotic conditions and in whole organ transplantation. Activation of the endothelial cells and shedding of the protective vascular glycocalyx during IRI increase the risk of innate immune activation, cell infiltration and severe thrombus formation, promoting damage to the tissue. Here, we present a novel one-step strategy to protect the vasculature by immobilisation of a unique multi-arm heparin conjugate to the endothelium. Applying a new in vitro blood endothelial cell chamber model, the heparin conjugate was found to bind not only to primary human endothelial cells but also directly to the collagen to which the cells adhered. Incubation of hypoxic endothelial cells with freshly drawn human blood in the blood chambers elicited coagulation activation reflected by thrombin anti-thrombin formation and binding of platelets and neutrophils. Immobilisation of the heparin conjugate to the hypoxic endothelial cells created a protective coating, leading to a significant reduction of the recruitment of blood cells and coagulation activation compared to untreated hypoxic endothelial cells. This novel approach of immobilising multi-arm heparin conjugates on the endothelial cells and collagen of the basement membrane ensures to protect the endothelium against IRI in thrombotic disorders and in transplantation.

Hormonal therapies targeting androgen receptor (AR) are effective in prostate cancer (PCa), but often the cancers progress to fatal castrate-resistant disease. Improved understanding of the cellular events during androgen deprivation would help to identify survival and stress pathways whose inhibition could synergize with androgen deprivation. Toward this aim, we performed an RNAi screen on 2,068 genes, including kinases, phosphatases, epigenetic enzymes and other druggable gene targets. High-content cell spot microarray (CSMA) screen was performed in VCaP cells in the presence and absence of androgens with detection of Ki67 and cleaved ADP-ribose polymerase (cPARP) as assays for cell proliferation and apoptosis. Thirty-nine candidate genes were identified, whose silencing inhibited proliferation or induced apoptosis of VCaP cells exclusively under androgen-deprived conditions. One of the candidates, HSPB (heat shock 27 kDa)-associated protein 1 (HSPBAP1), was confirmed to be highly expressed in tumor samples and its mRNA expression levels increased with the Gleason grade. We found that strong HSPBAP1 immunohistochemical staining (IHC) was associated with shorter disease-specific survival of PCa patients compared with negative to moderate staining. Furthermore, we demonstrate that HSPBAP1 interacts with AR in the nucleus of PCa cells specifically during androgen-deprived conditions, occupies chromatin at PSA/klk3 and TMPRSS2/tmprss2 enhancers and regulates their expression. In conclusion, we suggest that HSPBAP1 aids in sustaining cell viability by maintaining AR signaling during androgen-deprived conditions., (© 2014 UICC.)

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways activated during cardiovascular pathologies are, however, limited by the lack of robust and physiologically relevant methods. In order to model pathological vascular events we have developed an in vitro assay for studying the interaction between endothelium and whole blood. The assay consists of primary human endothelial cells, which are placed in contact with human whole blood. The method utilizes native blood with no or very little anticoagulant, enabling study of delicate interactions between molecular and cellular components present in a blood vessel. We investigated functionality of the assay by comparing activation of coagulation by different blood volumes incubated with or without human umbilical vein endothelial cells (HUVEC). Whereas a larger blood volume contributed to an increase in the formation of thrombin antithrombin (TAT) complexes, presence of HUVEC resulted in reduced activation of coagulation. Furthermore, we applied image analysis of leukocyte attachment to HUVEC stimulated with tumor necrosis factor (TNFα) and found the presence of CD16(+) cells to be significantly higher on TNFα stimulated cells as compared to unstimulated cells after blood contact. In conclusion, the assay may be applied to study vascular pathologies, where interactions between the endothelium and the blood compartment are perturbed.

Background: Radical prostatectomy reduces mortality among men with localized prostate cancer; however, important questions regarding long-term benefit remain., Methods: Between 1989 and 1999, we randomly assigned 695 men with early prostate cancer to watchful waiting or radical prostatectomy and followed them through the end of 2012. The primary end points in the Scandinavian Prostate Cancer Group Study Number 4 (SPCG-4) were death from any cause, death from prostate cancer, and the risk of metastases. Secondary end points included the initiation of androgen-deprivation therapy., Results: During 23.2 years of follow-up, 200 of 347 men in the surgery group and 247 of the 348 men in the watchful-waiting group died. Of the deaths, 63 in the surgery group and 99 in the watchful-waiting group were due to prostate cancer; the relative risk was 0.56 (95% confidence interval [CI], 0.41 to 0.77; P=0.001), and the absolute difference was 11.0 percentage points (95% CI, 4.5 to 17.5). The number needed to treat to prevent one death was 8. One man died after surgery in the radical-prostatectomy group. Androgen-deprivation therapy was used in fewer patients who underwent prostatectomy (a difference of 25.0 percentage points; 95% CI, 17.7 to 32.3). The benefit of surgery with respect to death from prostate cancer was largest in men younger than 65 years of age (relative risk, 0.45) and in those with intermediate-risk prostate cancer (relative risk, 0.38). However, radical prostatectomy was associated with a reduced risk of metastases among older men (relative risk, 0.68; P=0.04)., Conclusions: Extended follow-up confirmed a substantial reduction in mortality after radical prostatectomy; the number needed to treat to prevent one death continued to decrease when the treatment was modified according to age at diagnosis and tumor risk. A large proportion of long-term survivors in the watchful-waiting group have not required any palliative treatment. (Funded by the Swedish Cancer Society and others.).

Patients with long-standing inflammatory bowel disease (IBD) have an increased risk for colorectal carcinoma (CRC). Earlier studies suggest that the severity of inflammation is an independent risk factor for CRC in ulcerative colitis (UC). We investigated the role of histological inflammation as a risk factor for colorectal dysplasia or CRC to better target dysplasia surveillance in IBD. By combining our hospital patient registry and pathology database between 1996 and 2008, we identified 183 IBD patients with dysplasia or CRC. The control group was collected from our registry of IBD patients. Histological severe inflammation was present in 41.4% of patients with dysplasia and in 24.1% of patients with CRC, but in only 4.3% of controls. Severe inflammation had an odds ratio (OR) of 31.8 [95% confidence interval (CI): 15.6-64.9] for dysplasia or carcinoma compared to patients with no inflammation. Among patients with mild to moderate inflammation, the OR was 2.6 (95% CI: 1.6-4.1). Disease duration increased the annual risk for dysplasia or CRC by 4.5%. Coexisting primary sclerosing cholangitis (PSC) did not elevate the risk, whereas use of thiopurines (OR = 0.09, 95% CI: 0.02-0.33) and also 5-aminosalicylic acid (OR 0.17, 95% CI: 0.017-1.01) protected against CRC. As conclusion, degree of inflammation and duration of disease cumulatively increase the risk for dysplasia and CRC. PSC was not identified as a risk factor. We demonstrated that use of thiopurines strongly protects against CRC. These results can be applied to better target dysplasia surveillance in IBD patients., (© 2013 UICC.)

Background: PSA is the most useful prostate cancer marker. However, its levels are increased also in some non-malignant conditions. In circulation, the majority of PSA is complexed with protease inhibitors, including α(1) -antichymotrypsin (ACT). The proportion of the PSA-ACT complex is higher in patients with prostate cancer than in controls without cancer. The expression of ACT has been shown to be higher in prostate cancer than in benign prostatic hyperplasia. However, results regarding the extent which PSA forms complexes within the prostate and whether there are differences in complex formation between normal and malignant prostatic tissue are inconsistent and limited., Methods: We studied complex formation of PSA secreted by cultured human prostate tissues and in the tissue by in situ proximity ligation assay (PLA). Free, total and active PSA, and the PSA-ACT complex were determined in tissue culture media by immunoassays, immunoblotting, and chromatographic methods., Results: The majority of PSA in tissue culture medium was free and enzymatically active. However, a significant proportion (1.6 ± 0.5%) of immunoreactive PSA was found to be complexed with ACT. Complex formation was confirmed by in situ PLA, which showed more intense staining of PSA-ACT in cancers with Gleason grade 3 than in adjacent benign tissues from the same patients., Conclusions: These results show that PSA forms complexes already within the prostate and that PSA-ACT levels are increased in moderately differentiated prostate cancer tissue. This may explain, at least partially, why the ratio of serum PSA-ACT to total PSA is increased in prostate cancer., (Copyright © 2012 Wiley Periodicals, Inc.)

Background: Digital whole-slide scanning of tissue specimens produces large images demanding increasing storing capacity. To reduce the need of extensive data storage systems image files can be compressed and scaled down. The aim of this article is to study the effect of different levels of image compression and scaling on automated image analysis of immunohistochemical (IHC) stainings and automated tumor segmentation., Methods: Two tissue microarray (TMA) slides containing 800 samples of breast cancer tissue immunostained against Ki-67 protein and two TMA slides containing 144 samples of colorectal cancer immunostained against EGFR were digitized with a whole-slide scanner. The TMA images were JPEG2000 wavelet compressed with four compression ratios: lossless, and 1:12, 1:25 and 1:50 lossy compression. Each of the compressed breast cancer images was furthermore scaled down either to 1:1, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64 or 1:128. Breast cancer images were analyzed using an algorithm that quantitates the extent of staining in Ki-67 immunostained images, and EGFR immunostained colorectal cancer images were analyzed with an automated tumor segmentation algorithm. The automated tools were validated by comparing the results from losslessly compressed and non-scaled images with results from conventional visual assessments. Percentage agreement and kappa statistics were calculated between results from compressed and scaled images and results from lossless and non-scaled images., Results: Both of the studied image analysis methods showed good agreement between visual and automated results. In the automated IHC quantification, an agreement of over 98% and a kappa value of over 0.96 was observed between losslessly compressed and non-scaled images and combined compression ratios up to 1:50 and scaling down to 1:8. In automated tumor segmentation, an agreement of over 97% and a kappa value of over 0.93 was observed between losslessly compressed images and compression ratios up to 1:25., Conclusions: The results of this study suggest that images stored for assessment of the extent of immunohistochemical staining can be compressed and scaled significantly, and images of tumors to be segmented can be compressed without compromising computer-assisted analysis results using studied methods., Virtual Slides: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2442925476534995.

Background: The aim of the study was to assess whether texture analysis is feasible for automated identification of epithelium and stroma in digitized tumor tissue microarrays (TMAs). Texture analysis based on local binary patterns (LBP) has previously been used successfully in applications such as face recognition and industrial machine vision. TMAs with tissue samples from 643 patients with colorectal cancer were digitized using a whole slide scanner and areas representing epithelium and stroma were annotated in the images. Well-defined images of epithelium (n = 41) and stroma (n = 39) were used for training a support vector machine (SVM) classifier with LBP texture features and a contrast measure C (LBP/C) as input. We optimized the classifier on a validation set (n = 576) and then assessed its performance on an independent test set of images (n = 720). Finally, the performance of the LBP/C classifier was evaluated against classifiers based on Haralick texture features and Gabor filtered images., Results: The proposed approach using LPB/C texture features was able to correctly differentiate epithelium from stroma according to texture: the agreement between the classifier and the human observer was 97 per cent (kappa value = 0.934, P < 0.0001) and the accuracy (area under the ROC curve) of the LBP/C classifier was 0.995 (CI95% 0.991-0.998). The accuracy of the corresponding classifiers based on Haralick features and Gabor-filter images were 0.976 and 0.981 respectively., Conclusions: The method illustrates the capability of automated segmentation of epithelial and stromal tissue in TMAs based on texture features and an SVM classifier. Applications include tissue specific assessment of gene and protein expression, as well as computerized analysis of the tumor microenvironment., Virtual Slides: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/4123422336534537.

Prostate cancer is the second leading cause of cancer mortality in men in developed countries. Due to the heterogeneous nature of the disease, design of novel personalized treatments is required to achieve efficient therapeutic responses. We have recently identified phospholipase 2 group VII (PLA2G7) as a potential drug target especially in ERG oncogene positive prostate cancers. Here, the expression profile of PLA2G7 was studied in 1137 prostate cancer and 409 adjacent non-malignant prostate tissues using immunohistochemistry to validate its biomarker potential and putative association with disease progression. In order to reveal the molecular alterations induced by PLA2G7 impairment, lipidomic and gene expression profiling was performed in response to PLA2G7 silencing in cultured prostate cancer cells. Moreover, the antineoplastic effect of statins combined with PLA2G7 impairment was studied in prostate cancer cells to evaluate the potential of repositioning of in vivo compatible drugs developed for other indications towards anti-cancer purposes. The results indicated that PLA2G7 is a cancer-selective biomarker in 50 % of prostate cancers and associates with aggressive disease. The alterations induced by PLA2G7 silencing highlighted the potential of PLA2G7 inhibition as an anti-proliferative, pro-apoptotic and anti-migratorial therapeutic approach in prostate cancer. Moreover, the anti-proliferative effect of PLA2G7 silencing was potentiated by lipid-lowering statins in prostate cancer cells. Taken together, our results support the potential of PLA2G7 as a biomarker and a drug target in prostate cancer and present a rationale for combining PLA2G7 inhibition with the use of statins in prostate cancer management.

High androgen receptor (AR) level in primary tumour predicts increased prostate cancer-specific mortality. However, the mechanisms that regulate AR function in prostate cancer are poorly known. We report here a new paradigm for the forkhead protein FoxA1 action in androgen signalling. Besides pioneering the AR pathway, FoxA1 depletion elicited extensive redistribution of AR-binding sites (ARBs) on LNCaP-1F5 cell chromatin that was commensurate with changes in androgen-dependent gene expression signature. We identified three distinct classes of ARBs and androgen-responsive genes: (i) independent of FoxA1, (ii) pioneered by FoxA1 and (iii) masked by FoxA1 and functional upon FoxA1 depletion. FoxA1 depletion also reprogrammed AR binding in VCaP cells, and glucocorticoid receptor binding and glucocorticoid-dependent signalling in LNCaP-1F5 cells. Importantly, FoxA1 protein level in primary prostate tumour had significant association to disease outcome; high FoxA1 level was associated with poor prognosis, whereas low FoxA1 level, even in the presence of high AR expression, predicted good prognosis. The role of FoxA1 in androgen signalling and prostate cancer is distinctly different from that in oestrogen signalling and breast cancer.

Background: In 2008, we reported that radical prostatectomy, as compared with watchful waiting, reduces the rate of death from prostate cancer. After an additional 3 years of follow-up, we now report estimated 15-year results., Methods: From October 1989 through February 1999, we randomly assigned 695 men with early prostate cancer to watchful waiting or radical prostatectomy. Follow-up was complete through December 2009, with histopathological review of biopsy and radical-prostatectomy specimens and blinded evaluation of causes of death. Relative risks, with 95% confidence intervals, were estimated with the use of a Cox proportional-hazards model., Results: During a median of 12.8 years, 166 of the 347 men in the radical-prostatectomy group and 201 of the 348 in the watchful-waiting group died (P=0.007). In the case of 55 men assigned to surgery and 81 men assigned to watchful waiting, death was due to prostate cancer. This yielded a cumulative incidence of death from prostate cancer at 15 years of 14.6% and 20.7%, respectively (a difference of 6.1 percentage points; 95% confidence interval [CI], 0.2 to 12.0), and a relative risk with surgery of 0.62 (95% CI, 0.44 to 0.87; P=0.01). The survival benefit was similar before and after 9 years of follow-up, was observed also among men with low-risk prostate cancer, and was confined to men younger than 65 years of age. The number needed to treat to avert one death was 15 overall and 7 for men younger than 65 years of age. Among men who underwent radical prostatectomy, those with extracapsular tumor growth had a risk of death from prostate cancer that was 7 times that of men without extracapsular tumor growth (relative risk, 6.9; 95% CI, 2.6 to 18.4)., Conclusions: Radical prostatectomy was associated with a reduction in the rate of death from prostate cancer. Men with extracapsular tumor growth may benefit from adjuvant local or systemic treatment. (Funded by the Swedish Cancer Society and the National Institutes of Health.).

Matrix metalloproteinase-7 is capable of degrading many extracellular matrix proteins and cellular adhesions. In many malignancies, it is overexpressed, and it plays a role in cancer progression by enhancing tumor invasion and thereby metastatic potential. The purpose of this study was to evaluate the association between MMP-7 tissue expression and prognosis in colorectal cancer. From 623 patients who underwent surgery for colorectal cancer, surgical specimens were collected into tissue array blocks and stained by immunohistochemistry for MMP-7. Specimens from 545 patients were suitable for analysis. In specimens from 105 patients (19.3%), MMP-7 scored as high; in 103 (18.9%), as moderate; and in 134 (24.9%), as mild. In 203 cases (37.2%), immunoreactivity was negative. A significant correlation appeared between MMP-7 immunoexpression and tumor differentiation. High MMP-7 positivity associated with poor prognosis during a 5-year follow-up. During longer follow-up, the differences in survival between groups disappeared. MMP-7 is a potential target for tumor therapy, which should be evaluated in clinical trials.

To enable cancer to invade and to metastasize, the surrounding stroma must be degraded. Matrix metalloproteinase-7 (MMP-7) is capable of degrading many extracellular matrix proteins and cellular adhesions, is overexpressed in many malignancies, and plays a role in tumour progression. The purpose of this study was to evaluate the association between MMP-7 tissue expression and patients' prognosis in gastric cancer. From 264 patients who underwent surgery for gastric cancer, surgical specimens were collected on tissue array blocks and stained by immunohistochemistry for MMP-7. In 27 (10.2%) of the specimens, immunopositivity was found as high, in 50 (18.9%) as moderate and in 51 (19.3%) as weak. In 136 cases (51.5%), the immunopositivity was negative. A statistically significant correlation appeared between high MMP-7 expression and poor survival. In conclusion, our results suggest that MMP-7 expression may prove helpful in evaluating gastric cancer prognosis.

Cyclooxygenase-2 (COX-2) is an important factor in gastric carcinogenesis, and COX-2 expression in gastric cancer patients correlates with prognosis. We have now studied the impact of COX-2 in comparison to six other tissue tumor markers, DNA index, and S-phase fraction (SPF) in a large series of gastric cancer specimens. From 342 consecutive patients, 337 archival tissue specimens were available for immunohistochemistry of COX-2, HuR, cyclin A, MMP-2, p53, p21, and Ki-67 and 313 for analysis of DNA index and S-phase fraction by flow cytometry. Associations between factors were assessed by chi-square test and survival analysis by the Kaplan-Meier method and Cox model. A significant association emerged between of COX-2 and p53 (p < 0.0001), Ki-67 (p = 0.013), DNA ploidy (p < 0.0001), and SPF (p < 0.0001). In an extended multivariate analysis, COX-2 and p53 expression were independent prognostic factors for poor survival, in addition to high stage and non-curative surgery. In gastric cancer, COX-2 expression associated with markers for apoptosis and proliferation, and furthermore, it was confirmed that COX-2 and p53 are strong prognostic indicators.

Xanthine oxidoreductase (XOR) is a key enzyme in degradation of DNA and RNA, and has previously been shown to be decreased in aggressive breast and gastric cancer. In this study, XOR expression was assessed in tissue microarray specimens of 478 patients with colorectal cancer and related to clinical parameters. In addition, we performed in vitro studies of XOR activity, protein and mRNA in colon cancer cells (Caco-2). Results from the tissue expression analyses show that XOR was decreased in 62% and undetectable in 22% of the tumours as compared to normal tissue. Loss of XOR was associated with poor grade of differentiation (p=0.006) and advanced Dukes stage (p=0.03). In multivariate survival analysis, XOR was a prognostic factor (p=0.008), independent of Dukes stage, histological grade, age and tumour location. The in vitro analyses show that XOR is not measurable in undifferentiated Caco-2 cells, but appears and increases with differentiation. We conclude that XOR expression is associated with histological grade of differentiation and extent of disease in colorectal cancer, and it provides significant prognostic information independently of established factors.

Objectives: To analyze the expression of the androgen receptor(AR) and syndecan-1 in breast tissue during long-term hormonal treatment in cynomolgus monkeys., Methods: Sixty oophorectomized macaques were randomized to receive either tibolone, conjugated equine estrogens (CEE), CEE + medroxyprogesterone acetate (MPA) or no hormonal treatment. Breast tissue was collected at necropsy after 2 years and stained for AR and syndecan-1., Results: Apparent differences were seen between treatment groups as compared to untreated animals. AR expression was markedly increased by tibolone and suppressed by combined CEE/MPA. Both treatments increased syndecan-1 in stromal tissue, whereas CEE alone had no significant effect., Conclusions: We found alternative regimens for hormonal therapy to differ in their influence on two markers of importance for the development of breast cancer. The results may be relevant for the ongoing clinical discussion on the long-term safety of different hormonal treatments.

Background: The benefit of radical prostatectomy in patients with early prostate cancer has been assessed in only one randomized trial. In 2005, we reported that radical prostatectomy improved prostate cancer survival compared with watchful waiting after a median of 8.2 years of follow-up. We now report results after 3 more years of follow-up., Methods: From October 1, 1989, through February 28, 1999, 695 men with clinically localized prostate cancer were randomly assigned to radical prostatectomy (n = 347) or watchful waiting (n = 348). Follow-up was complete through December 31, 2006, with histopathologic review and blinded evaluation of causes of death. Relative risks (RRs) were estimated using the Cox proportional hazards model. Statistical tests were two-sided., Results: During a median of 10.8 years of follow-up (range = 3 weeks to 17.2 years), 137 men in the surgery group and 156 in the watchful waiting group died (P = .09). For 47 of the 347 men (13.5%) who were randomly assigned to surgery and 68 of the 348 men (19.5%) who were not, death was due to prostate cancer. The difference in cumulative incidence of death due to prostate cancer remained stable after about 10 years of follow-up. At 12 years, 12.5% of the surgery group and 17.9% of the watchful waiting group had died of prostate cancer (difference = 5.4%, 95% confidence interval [CI] = 0.2 to 11.1%), for a relative risk of 0.65 (95% CI = 0.45 to 0.94; P = .03). The difference in cumulative incidence of distant metastases did not increase beyond 10 years of follow-up. At 12 years, 19.3% of men in the surgery group and 26% of men in the watchful waiting group had been diagnosed with distant metastases (difference = 6.7%, 95% CI = 0.2 to 13.2%), for a relative risk of 0.65 (95% CI = 0.47 to 0.88; P = .006). Among men who underwent radical prostatectomy, those with extracapsular tumor growth had 14 times the risk of prostate cancer death as those without it (RR = 14.2, 95% CI = 3.3 to 61.8; P < .001)., Conclusion: Radical prostatectomy reduces prostate cancer mortality and risk of metastases with little or no further increase in benefit 10 or more years after surgery.

Aim: The purpose of this study was to assess if the addition of a panel of tumor markers to established clinicopathological factors could improve the accuracy of 5-year outcome prediction in gastric cancer. The studied markers were chosen to represent different aspects of tumor biology., Patients and Methods: The expression of syndecan-1, tenascin-C, tumor-associated trypsin inhibitor (TATI), p53, p21 and bcl-2 was analyzed by immunohistochemistry informalin-fixed, paraffin-embedded specimens from 337 patients with gastric cancer. In addition, the DNA ploidy and S-phase fraction (SPF) were assessed by flow cytometry., Results: The loss of epithelial syndecan-1, strong stromal syndecan-1, the loss of stromal tenascin-C, the loss of tumor tissue TATI, high p53 and high p21 expression, aneuploidy and high (> or =7.6%) SPF were all associated with an unfavorable prognosis in univariate survival analysis. In multivariate survival analysis, p53 (hazard ratio (HR) 1.58; confidence interval (CI) 95% 1.16-2.16), p21 (HR 1.67; CI 95% 1.09-2.56) and DNA ploidy (HR 1.50; CI 95% 1.10-2.05) were independent prognostic factors, in addition to penetration depth (pT), lymph node status (pN), age at diagnosis and estimated radicality of surgery. The difference in prognostic accuracy between a base model with pT, pN, age and radicality of surgery (area under the curve (AUC) 0.898; CI 95% 0.86-0.94) and an extended model including p53, p21 and DNA ploidy (AUC 0.900; CI 95% 0.86-0.94) was not statistically significant (p=0.85)., Conclusion: In gastric cancer, p53 and p21 expression, as well as DNA ploidy, are independent prognostic factors in addition to standard clinicopathological factors. However, the established indicators of the extent of disease show an impressively high accuracy in 5-year outcome prediction and adding the examined tumor markers to the base model does not significantly improve the prognostic accuracy.

Objective: The extracellular matrix glycoprotein tenascin-C has been proposed as a tumor marker with prognostic significance in many cancer forms, but in colorectal cancer, reported results have been controversial. The aim of this study was to evaluate the prognostic value of immunohistochemical tenascin-C expression in a series of 231 patients with colorectal cancer., Methods: Paraffin-embedded formalin-fixed specimens were stained with a tenascin-C-specific monoclonal antibody, and the stromal staining intensity and pattern were analyzed., Results: Tenascin-C immunoreactivity was observed in all 231 specimens, with a pattern of staining that was diffuse and interstitial. The staining was occasional in 39 (17%), moderate in 106 (46%) and strong in 86 specimens (37%). There was no statistically significant association between tenascin-C immunoreactivity and any of the other clinicopathological variables. The cumulative 5-year survival rates of patients with occasional and weak staining were similar (56.8 and 54.9%, respectively), while the patients with strong tenascin-C staining had a lower survival rate (46.1%). This difference in survival was not significant (p = 0.23). The staining pattern and distribution can be viewed from digitized representative microscope slides (virtual slides) at http://www.webmicroscope.net/supplements/tenascin., Conclusions: Our results indicate that immunohistochemical expression of tenascin-C is not of prognostic significance in colorectal cancer., ((c) 2008 S. Karger AG, Basel)

Background: The aim of the present study was to evaluate a series of biomarkers with regard to long-term prognostic value in patients with T1 (< or =2 cm) node-negative breast cancer., Method: The prognostic value of Ki-67, p53, oestrogen receptor (ER) immunohistochemical labelling, flow-cytometric S phase fraction and ploidy was evaluated in 212 patients with pT1N0M0 breast cancer. The median follow-up time was 15.9 years (range 0.2-27.2 years)., Results: In an analysis of breast cancer-specific survival up to 5 years, high Ki-67 (> or =10%; p = 0.002), high p53 (> or =20%; p = 0.01), negative ER (<30%; p = 0.01) as well as aneuploidy of the tumour (p = 0.02) were significant prognostic factors. When the follow-up was extended to 10 years, only Ki-67 (p = 0.03) was significantly associated with outcome and beyond 15 years none of the studied markers provided significant prognostic information when analyzed separately. There was a weak but significant difference in long-term survival when patients with a combination of high Ki-67 (> or =10%), high SPF (>3%) and high p53 (> or =20%) were compared to patients with other combinations (p = 0.03)., Conclusion: According to the results of our series, it seems that several prognostic markers which are associated with short-term survival (< or =5 years) in pT1N0M0 breast cancer may not be significant predictors of long-term (>15 years) breast cancer-specific survival.

High level of cyclin A promotes carcinogenesis, and overexpression of cyclin A has been associated with poor prognosis of cancer patients. We validated the prognostic role of cyclin A in gastric cancer and evaluated its correlation with expression of an mRNA stability factor HuR. From 342 consecutive histologically confirmed gastric cancer patients were obtained 325 representative tissue specimens for cyclin A and 316 for HuR immunohistochemistry. Specimens were stained by cyclin A and HuR specific monoclonal antibodies. Nuclear immunostaining detected in > or =5% of the tumor cells was considered the cut-off for cyclin A positivity. Positive HuR immunoreactivity was scored as nuclear or cytoplasmic. Associations between scores, clinicopathological factors and survival were calculated by the chi2-test, Fisher's exact test, Kaplan-Meier test and Cox model. Cyclin A detected in the nuclei of cancer cells was positive in 55% (179 of 325) of the specimens; 40% (127 of 316) of the specimens had cytoplasmic and 88% (279 of 316) nuclear immunoreactivity of HuR. Cyclin A expression was an independent prognostic factor for poor survival. Cyclin A immunoreactivity was associated with old age, high stage, proximal location of the tumor, intestinal type, noncurative resection, advanced penetration depth and with nodal metastases but not distant metastases. Furthermore, cyclin A expression was associated with cytoplasmic HuR expression, whereas no association with nuclear HuR was evident. Cyclin A is an independent prognostic factor in gastric cancer, and one mechanism for its overexpression may depend on cytoplasmic localization of HuR., (Copyright 2006 Wiley-Liss, Inc.)

Background: Xanthine oxidoreductase (XOR) is a key enzyme in the degradation of DNA, RNA and high-energy phosphates. About half of the patients with breast cancer have a decrease in XOR expression. Patients with breast cancer with unfavourable prognosis are independently identified by the loss of XOR., Aim: To assess the clinical relevance of XOR expression in gastric cancer., Methods: XOR levels were studied by immunohistochemistry in tissue microarray specimens of 337 patients with gastric cancer and the relation between XOR expression and a series of clinicopathological variables, as well as disease-specific survival, was assessed., Results: XOR was moderately decreased in 41% and was undetectable in another 14% of the tumours compared with the corresponding normal tissue. Decreased XOR was associated with advanced stage, deep tumour penetration, diffusely spread tumour location, positive lymph node status, large tumour size, non-curative disease, cellular aneuploidy, high S-phase fraction and high cyclooxygenase-2 expression, but not with p53 expression or Borrmann classification. Down regulation of XOR was associated with unfavourable outcome, and the cumulative 5-year gastric cancer-specific survival in patients with strong XOR expression was 47%, compared with 22% in those with moderate to negative expression (p<0.001). This was also true in patients with stage I-II (p = 0.01) and lymph node-negative (p = 0.02) disease, as well as in patients with smaller (< or =5 cm) tumours (p = 0.02)., Conclusion: XOR expression in gastric cancer may be a new marker for a more aggressive gastric cancer biology, similar to that previously reported for breast cancer.

Background and Aim: Matrix metalloproteinases (MMPs) MMP-2 and MMP-9 can degrade type IV collagen of extracellular matrix and basal membranes. As cyclo-oxygenase-2 (COX-2) has been shown to activate MMPs, creating one of the COX-2-promoted pathways of tumour growth and metastasis, the prognostic role of MMP-2 and MMP-9 in gastric cancer was assessed and their association with COX-2 expression was evaluated., Materials and Methods: Samples were collected from 342 consecutive patients operated on for gastric cancer, of which 315 were acceptable for MMP-2, MMP-9 and COX-2 immunohistochemistry. Specimens were stained with specific antibodies, evaluated and categorised by two interpreters, and then correlated with clinical data and survival., Results: Epithelial MMP-2 immunoreactivity was associated with male sex, high stage, advanced penetration depth, non-curative surgery, high COX-2 expression and poor survival. Stromal MMP-2 expression correlated with high stage, intestinal type and non-curative surgery whereas MMP-9 correlated only with intestinal type. Stage, intent of surgery and COX-2 were independent prognostic factors., Conclusions: Epithelial MMP-2 expression in gastric cancer is associated with aggressive forms, COX-2 and poor survival, although MMP-2 was not an independent prognostic factor. In gastric cancer tumour growth is apparently induced by COX-2, and invasion is mediated by MMP-2.

In papillary thyroid cancer (PTC), age appears to be the most important single prognostic factor. Another characteristic feature is the lack of association between survival and lymph node metastases. Earlier, we found that expression of cyclooxygenase-2 (COX-2) is higher in older PTC patients, in agreement with the finding that older patients have a worse prognosis. Recent findings suggest that COX-2 can up-regulate vascular endothelial growth factor-C (VEGF-C) expression. Here, we investigated whether expression of VEGF-C differs between young and older PTC patients and whether expression of VEGF-C and COX-2 are correlated. Our retrospective study comprised 106 PTC patients selected by age: those under 35 or over 55 at diagnosis. Paraffin-embedded tissue samples were analysed by immunohistochemistry for VEGF-C protein expression. Furthermore, we investigated by quantitative RT-PCR and enzyme immunoassay the relationship between VEGF-C and COX-2 expression in papillary thyroid cancer cells (NPA cells). VEGF-C expression was significantly increased with age. In the tumours from older lymph node-positive (N1) patients, VEGF-C expression was significantly higher than in the tumours from young N1 patients. Moreover, all patients who died of cancer or who developed distant metastases were old, and most tumours from these patients (4 of 5) expressed VEGF-C and had had nodal metastases at the time of primary operation. Immunohistochemically, expression of COX-2 and VEGF-C correlated strongly. In cell culture, this correlation was not so clear, because the COX-2 selective inhibitor, NS-398, did not reduce VEGF-C expression. However, as both COX-2 and VEGF-C were induced by the tumour promoter phorbol 12-myristate 13-acetate (PMA), the same factors may control them both.

Background: Cyclooxygenase-2 (COX-2) overexpression is related to poor outcome in several cancers. COX-2 is upregulated in 42-90% of pancreatic ductal adenocarcinomas and is a potential target for chemotherapy. Earlier studies have not shown the expression of COX-2 to be a prognostic factor in pancreatic cancer., Objective: To evaluate the prognostic value of COX-2 in a series of patients with pancreatic adenocarcinoma., Methods: 128 patients operated on for pancreatic adenocarcinoma at Helsinki University Central Hospital between 1974 and 1998 provided sections from primary tumours which were immunohistochemically stained with a COX-2-antihuman monoclonal antibody., Results: Cytoplasmic COX-2 reactivity (>5%) occurred in 46 specimens (36%), correlating neither with age, sex, stage, size, tumour stage, nodal metastases, nor grade. Lack of COX-2 expression correlated with distant metastases (p = 0.026). In univariate survival analysis, COX-2 expression (p = 0.0114), stage (p = 0.0002), grade (p = 0.0001), and age (p = 0.042) had prognostic significance. One, two, and five year survival rates were 51%, 32%, and 8% in the COX-2 negative groups compared with 34%, 5%, and 5% in the COX-2 positive groups (p = 0.011). Prognostic significance was especially high for patients operated on with curative intent (p = 0.004). In multivariate analysis, COX-2 was an independent prognostic factor (hazard ratio = 1.6 (95% confidence interval, 1.1 to 2.3))., Conclusions: Expression of COX-2 was associated with poor outcome from pancreatic ductal adenocarcinoma and was independent of tumour stage, grade, or age in multivariate analysis.

Objective: Matrix metalloproteinases (MMPs) degrade extracellular matrix and are involved in tumor invasion and metastasis in various cancers. In pancreatic cancer, MMP-2 expression is upregulated and correlates with tumor recurrence. The aim of this study was to evaluate the prognostic significance of MMP-2 in pancreatic ductal adenocarcinoma., Methods: MMP-2 expression was assessed by immunohistochemistry in 127 patients operated on at Helsinki University Hospital from 1974 to 1998, with expression interpreted separately in epithelial and stromal samples., Results: Epithelial MMP-2 expression was strong in 5%, moderate in 20%, weak in 25% and negative in 50% of the tumors, with high epithelial MMP-2 expression significantly associated in univariate survival analysis with advanced stage, poor grade and poor survival. Stromal MMP-2 expression was strong in 0%, moderate in 14%, weak in 70% and negative in 16% of the cases, and did not significantly correlate with patient survival., Conclusion: Epithelial MMP-2 correlates with advanced tumor stage and grade, but is not an independent predictor of survival.

Purpose: Cyclooxygenase-2 (COX-2) promotes carcinogenesis and its expression associates with clinicopathologic characteristics in gastric cancer. HuR is an mRNA binding protein that controls the stability of certain transcripts including COX-2. We evaluated the prognostic significance of COX-2 and HuR expressions in gastric cancer and whether there exists a link between HuR and COX-2 expressions., Experimental Design: The study included 342 consecutive patients with histologically confirmed gastric adenocarcinoma, of whom 321 patients had tissue specimens available for COX-2 and 316 for HuR immunohistochemistry. Specimens were stained by COX-2- and HuR-specific monoclonal antibodies and scored by two independent observers. Correlation to clinical data and survival was assessed. TMK-1 gastric adenocarcinoma cells were treated with small interfering RNA against HuR and expressions of HuR and COX-2 were detected by immunofluorescence and Western blot analysis., Results: Patients with low COX-2 expression had a cumulative 5-year survival of 53% and those with high COX-2 expression had 16% (P < 0.0001). In multivariate analysis, COX-2 was an independent prognostic factor (P = 0.003). Cytoplasmic HuR expression was associated with high COX-2 expression (P < 0.0001) and with reduced survival (P = 0.004) whereas nuclear positivity for HuR was not. When TMK-1 cells were treated with HuR small interfering RNA, expressions of HuR and COX-2 were reduced., Conclusions: High COX-2 is an independent prognostic factor in gastric cancer. Cytoplasmic expression of HuR associates with high COX-2 expression and with reduced survival, and tissue culture experiments show that HuR can regulate expression of COX-2 in gastric cancer cells.

Background: Prostate-specific antigen (PSA/KLK3) and human kallikrein 2 (hK2/KLK2) belong to the human kallikrein gene family. These two highly homologous genes are specifically expressed in the prostate under androgen control. Expression of these is regulated by similar mechanisms but changes in their relative expression have been observed in prostate cancer., Methods: We determined the relative levels of PSA and hK2 mRNA in benign and malignant prostate tissue using a quantitative reverse transcription-polymerase chain reaction (RT-PCR) method. The mRNA of PSA and hK2 are reverse transcribed and amplified in one reaction with the same primers., Results: The variation in the ratio of hK2/PSA mRNA was remarkably small, the difference between the highest and lowest values being three-fold. The ratio was significantly higher in WHO grade 2 compared to normal or benign prostatic hyperplasia tissue (P = 0.032 and P = 0.035, respectively) and in grade 3 compared to normal or benign prostatic hyperplasia tissue (P = 0.006 in both)., Conclusions: The new quantitative RT-PCR technique facilitates very accurate quantitation of the relative mRNA levels of homologous genes. Using this method we have shown that the ratio of hK2/PSA mRNA is higher in cancerous than in benign prostatic tissue., (Copyright 2004 Wiley-Liss, Inc.)

Background: In 2002, we reported the initial results of a trial comparing radical prostatectomy with watchful waiting in the management of early prostate cancer. After three more years of follow-up, we report estimated 10-year results., Methods: From October 1989 through February 1999, 695 men with early prostate cancer (mean age, 64.7 years) were randomly assigned to radical prostatectomy (347 men) or watchful waiting (348 men). The follow-up was complete through 2003, with blinded evaluation of the causes of death. The primary end point was death due to prostate cancer; the secondary end points were death from any cause, metastasis, and local progression., Results: During a median of 8.2 years of follow-up, 83 men in the surgery group and 106 men in the watchful-waiting group died (P=0.04). In 30 of the 347 men assigned to surgery (8.6 percent) and 50 of the 348 men assigned to watchful waiting (14.4 percent), death was due to prostate cancer. The difference in the cumulative incidence of death due to prostate cancer increased from 2.0 percentage points after 5 years to 5.3 percentage points after 10 years, for a relative risk of 0.56 (95 percent confidence interval, 0.36 to 0.88; P=0.01 by Gray's test). For distant metastasis, the corresponding increase was from 1.7 to 10.2 percentage points, for a relative risk in the surgery group of 0.60 (95 percent confidence interval, 0.42 to 0.86; P=0.004 by Gray's test), and for local progression, the increase was from 19.1 to 25.1 percentage points, for a relative risk of 0.33 (95 percent confidence interval, 0.25 to 0.44; P<0.001 by Gray's test)., Conclusions: Radical prostatectomy reduces disease-specific mortality, overall mortality, and the risks of metastasis and local progression. The absolute reduction in the risk of death after 10 years is small, but the reductions in the risks of metastasis and local tumor progression are substantial., (Copyright 2005 Massachusetts Medical Society.)