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Background: After radical prostatectomy (RP) for prostate cancer (PC), p53 alterations predict biochemical relapse (BCR), however, recent evidence suggests that metastatic relapse (MR) not BCR is a surrogate for PC specific mortality (PCSM). This updated analysis of a previously published study investigated the association between p53 aberrations, MR and PCSM in men with localised PC., Methods: Two hundred and seventy-one men with localised PC treated with RP were included. RP specimens stained for p53 by immunohistochemistry were scored as (a) percentage of p53-positive tumour nuclei; and (b) clustering, where ≥12 p53-positive cells within a ×200 power field was deemed 'cluster positive'. Associations between p53 status and clinical outcomes (BCR, MR and PCSM) were evaluated., Results: Increasing percentage of p53-positive nuclei was significantly associated with shorter time to BCR, MR and PCSM (All p < 0.001). Half of the patients were p53 cluster positive. p53 cluster positivity was significantly associated with poorer outcomes at all clinical endpoints (BCR: HR 2.0, 95% CI 1.51-2.65, p < 0.001; MR: HR 4.1, 95% CI 2.02-8.14, p < 0.001; PCSM: HR 12.2, 95% CI 1.6-93; p = 0.016). These associations were independent of other established prognostic variables., Conclusions: p53 aberrations in radical prostatectomy tissue predict clinically relevant endpoints of MR and PCSM.

Incidence and mortality from cancer is increasing in most countries in the world, with the highest burden in developing countries. City Cancer Challenge (C/Can), an initiative launched in 2017, aims to improve access to quality cancer care in metropolitan areas (1 million inhabitants or more) in low- and upper-middle income countries by transforming the way stakeholders at the city, regional, and national levels collectively design, plan, and implement local cancer solutions. The approach is built on the core principle that local leaders in cities define their own needs and craft solutions with the support of a network of global, regional, and local partners that reflect an understanding of the unique local context. C/Can aims to build a collective movement of cities that can together deliver quality, equitable, and sustainable cancer control solutions for all.

Background: Prostate cancers (PCs) with similar characteristics at the time of diagnosis can have very different disease outcomes. Conventional biomarkers of PC still lack precision in identifying individuals at high risk of PC recurrence. While many candidate biomarkers are proposed in the literature, few are in clinical practice as they lack rigorous validation. This study prospectively enrolled an independent phase III cohort to evaluate the clinical utility of zinc-alpha 2-glycoprotein (AZGP1) as a prognostic biomarker in localized PC., Patients and Methods: In our multicentre, prospective phase III study, AZGP1 status in 347 radical prostatectomy specimens was assayed by immunohistochemistry in a NATA-accredited laboratory. The AZGP1 score was assessed in a multivariable model incorporating established prognostic factors. We also report extended outcomes from our previous phase II study. The primary endpoint was biochemical relapse-free survival (BRFS). Secondary endpoints were metastasis-free survival (MFS) and PC-specific survival (PCSS)., Results: In the phase II cohort, with a median follow-up of 15.8 years, low/absent AZGP1 expression was an independent predictor of poor BRFS (HR, 1.4; 95% CI, 1.1-1.9; P = 0.03), MFS (HR, 2.8; 95% CI, 1.2-6.6; P = 0.02) and PCSS (HR, 3.8; 95% CI, 1.5-9.5; P = 0.005). These results were validated in our prospective phase III cohort. Low/absent AZGP1 expression independently predicted for BRFS (HR, 1.9; 95% CI, 1.1-3.3; P = 0.02), with shorter MFS (HR, 2.0; 95% CI, 1.1-3.4; P = 0.02). AZGP1 improved the discriminatory value when incorporated into existing prognostic risk models., Conclusion: Our study provides prospective phase III validation that absent/low AZGP1 expression provides independent prognostic value in PC. This study provides robust evidence for the incorporation of this biomarker into clinical practice., (© The Author 2017. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Despite incremental advances in the diagnosis and treatment for pancreatic cancer (PC), the 5‑year survival rate remains <5%. Novel therapies to increase survival and quality of life for PC patients are desperately needed. Epigenetic thera-peutic agents such as histone deacetylase inhibitors (HDACi) and DNA methyltransferase inhibitors (DNMTi) have demonstrated therapeutic benefits in human cancer. We assessed the efficacy of these epigenetic therapeutic agents as potential therapies for PC using in vitro and in vivo models. Treatment with HDACi [suberoylanilide hydroxamic acid (SAHA)] and DNMTi [5‑AZA‑2' deoxycytidine (5‑AZA‑dc)] decreased cell proliferation in MiaPaCa2 cells, and SAHA treatment, with or without 5‑AZA‑dc, resulted in higher cell death and lower DNA synthesis compared to 5‑AZA‑dc alone and controls (DMSO). Further, combination treatment with SAHA and 5‑AZA‑dc significantly increased expression of p21WAF1, leading to G1 arrest. Treatment with epigenetic agents delayed tumour growth in vivo, but did not decrease growth of established pancreatic tumours. In conclusion, these data demonstrate a potential role for epigenetic modifier drugs for the management of PC, specifically in the chemoprevention of PC, in combination with other chemotherapeutic agents.

Background: Docetaxel improves symptoms and survival in metastatic castration-resistant prostate cancer (CRPC). However, ∼50% of patients are chemoresistant. This study examined whether changes in cytokine levels predict for docetaxel resistance in vitro and in a clinical cohort., Methods: PC3 cells or their docetaxel-resistant subline (PC3Rx) were co-cultured with U937 monocytes, with and without docetaxel treatment, and cytokine levels were measured. The circulating levels of 28 cytokines were measured pre-/post cycle 1 of docetaxel from 55 men with CRPC, and compared with prostate-specific antigen (PSA) response., Results: PC3Rx-U937 co-culture expressed more cytokines, chiefly markers of alternative macrophage differentiation, compared with PC3-U937 co-culture. Docetaxel treatment enhanced cytokine production by PC3Rx-U937 co-culture, while reducing cytokine levels in PC3-U937. In patients, changes in the levels of seven circulating cytokines (macrophage inhibitory cytokine 1 (MIC1), interleukin (IL)-1ra, IL-1β, IL-4, IL-6, IL-12 and IFNγ) after cycle 1 of docetaxel were associated with progressive disease (all P<0.05). The combination of changes in MIC1, IL-4 and IL-6 most strongly predicted PSA response (P=0.002)., Conclusions: In vitro studies suggest docetaxel resistance is mediated, at least in part, by cytokines induced by the interaction between the docetaxel-resistant tumour cells and macrophages. Early changes in circulating cytokine levels were associated with docetaxel resistance in CRPC patients. When considered together, these data suggest a significant role for the inflammatory response and macrophages in the development of docetaxel resistance in CRPC.

Background: The PI3K pathway plays a significant role in the progression of prostate cancer (PCa) to an advanced stage. Mouse models suggest that the downstream effector molecule of the PI3K pathway, mTOR, is also important in the development of PCa, where it plays a pivotal role in forming precursor lesions such as high grade prostatic intraepithelial neoplasia (HGPIN). This study was conducted to determine the status of phosphorylated-mTOR (p-mTOR the activated state of mTOR) across the PCa progression model by looking at expression in normal prostate tissue, proliferative inflammatory atrophy (PIA), HGPIN, and PCa., Methods: Expression of p-mTOR was evaluated by immunohistochemistry on tissue microarrays constructed from 120 archival formalin-fixed paraffin embedded radical prostatectomy tissue specimens. Levels of expression were recorded as the percentage of positive epithelial cells multiplied by the intensity of staining scored as 0-3., Results: p-mTOR expression was found to increase across the progression model with mean staining in non-neoplastic samples of 40 compared to 98 in PIA, 107 in HGPIN, and 136 in cancer (P < 0.001), but without significant increase between HGPIN and PIA. Correlation of high p-mTOR expression with outcome in PCa showed a trend towards worse prognosis, but this was not statistically significant., Conclusions: This study demonstrates that p-mTOR signaling has a potential role in both the initiation and progression of PCa. These data provide support for further research into the possible use of rapamycin analogues in the treatment of PCa, and raise the possibility that mTOR might be a potential target for chemoprevention., (© 2014 Wiley Periodicals, Inc.)

Unlabelled: Study Type - Prognosis (case series) Level of Evidence 4 What's known on the subject? and What does the study add? Only 30-35% of patients with positive surgical margins after radical prostatectomy develop recurrent disease. Adjuvant radiotherapy reduces the rate of biochemical relapse or metastasis and improves overall survival after radical prostatectomy. Various pathological factors, such as location and extent of positive margins, have been proposed as possible prognostic factors in men with margin-positive prostate cancer, however, the recent International Society of Urological Pathology consensus meeting in Boston noted that there is limited data on the significance of Gleason grade of the carcinoma at a positive margin. The present study shows that the presence of high grade prostate cancer, i.e. Gleason pattern 4 or 5, at a positive surgical margin is an independent predictor of biochemical recurrence after radical prostatectomy. Moreover, patients with lower grade carcinoma at the margin have a similar prognosis to men with negative margins. Hence, assessment of Gleason grade at the site of positive margin may aid optimal selection of patients for adjuvant radiotherapy., Objective: • To establish predictors of biochemical recurrence by analysing the pathological characteristics of positive surgical margins (PSMs), including Gleason grade of the carcinoma at the involved margin., Patients and Methods: • Clinicopathological and outcome data on 940 patients who underwent radical prostatectomy (RP) between 1997 and 2003 were collected. • Of these, 285 (30.3%) patients with PSMs were identified for pathological review, including assessment of location of margin, linear extent, number of PSMs, plane of margin and Gleason grade (3 vs 4 or 5) at the margin., Results: • At a median follow-up of 82 months, the biochemical recurrence rate of the PSM cohort was 29%. • On univariate analysis, the presence of Gleason grade 4 or 5 at the margin (34.4% of cases) was significantly associated with biochemical recurrence (hazard ratio [HR] 2.80, 95% confidence interval [CI]= 1.82-4.32, P < 0.001) compared with the presence of Gleason grade 3. • Linear extent of margin involvement was also associated with recurrence (P= 0.009). • Single vs multiple margin involvement, location, and plane of the involved margin were not significant predictors of recurrence. • On multivariate analysis, Gleason grade 4 or 5 at the margin remained an independent predictor of recurrence (HR 2.14, 95% CI = 1.29-4.03, P= 0.003)., Conclusion: • The Gleason grade at the site of a PSM identifies patients at increased risk of biochemical recurrence and should aid stratification of patients for adjuvant radiation therapy., (© 2011 NHMRC CLINICAL TRIALS CENTRE. BJU INTERNATIONAL © 2011 BJU INTERNATIONAL.)

Background: Men with positive margins after radical prostatectomy (RP) for localized prostate cancer (PC) have a 40-50% biochemical relapse rate at 5 years. Adjuvant radiotherapy improves biochemical progression-free and overall survival in men with positive margins, but is associated with increased toxicity. There is an urgent need to identify new prognostic markers to define the group of patients who would benefit from multimodality therapy., Methods: Nuclear β-catenin, membranous secreted frizzled-related protein 4 (sFRP4), zinc-alpha 2-glycoprotein (AZGP1), and macrophage inhibitory cytokine-1 (MIC-1) have previously been identified as molecular markers of outcome in localized PC. From these published studies, we identified a subset of patients with positive margins. The aim of this study was to assess the association between these four molecular markers and outcome in men with margin-positive, localized PC., Results: We identified 186 men with positive margins from 330 men with localized PC; 53% had preoperative PSA >10 ng/ml, 72% extraprostatic extension (EPE), 24% seminal vesicles involvement (SVI), and 57% RP Gleason score ≥ 7. AZGP1 (P = 0.009), membranous sFRP4 (P = 0.03) and MIC-1 (P = 0.04) expression predicted for biochemical relapse on univariate analysis. Only absent/low AZGP1 expression (P = 0.01) was an independent predictor of recurrence in margin-positive, localized PC when modeled with preoperative PSA (P = 0.2), EPE (P = 0.2), SVI (P = 0.4), Gleason score ≥ 7 (P = 0.5) and adjuvant treatment (P = 0.4). Furthermore, there was an association between absent/low AZGP1 expression and clinical recurrence (P = 0.007)., Conclusions: AZGP1 is a potential molecular marker for biochemical relapse in men with margin-positive, localized PC. Routine assessment of this biomarker may lead to better selection of patients who will benefit from post-RP radiotherapy., (Copyright © 2011 Wiley-Liss, Inc.)

Chemotherapy remains the major treatment option for castration-resistant prostate cancer (CRPC) and limited cytotoxic options are available. Inherent chemotherapy resistance occurs in half of all patients and inevitably develops even in those who initially respond. Docetaxel has been the mainstay of therapy for 6 years, providing a small survival benefit at the cost of significant toxicity. Cabazitaxel is a promising second-line agent; however, it is no less toxic, whereas mitoxantrone provides only symptomatic benefit. Multiple cellular pathways involving apoptosis, inflammation, angiogenesis, signalling intermediaries, drug efflux pumps and tubulin are implicated in the development of chemoresistance. A thorough understanding of these pathways is needed to identify biomarkers that predict chemotherapy resistance with the aim to avoid unwarranted toxicities in patients who will not benefit from treatment. Until recently, the search for predictive biomarkers has been disappointing; however, the recent discovery of macrophage inhibitory cytokine 1 as a marker of chemoresistance may herald a new era of biomarker discovery in CRPC. Understanding the interface between this complex array of chemoresistance pathways rather than their study in isolation will be required to effectively predict response and target the late stages of advanced disease. The pre-clinical evidence for these resistance pathways and their progress through clinical trials as therapeutic targets is reviewed in this study.

Background: Previously, we showed that gene suppression commonly occurs across chromosome 2q14.2 in colorectal cancer, through a process of long-range epigenetic silencing (LRES), involving a combination of DNA methylation and repressive histone modifications. We now investigate whether LRES also occurs in prostate cancer across this 4-Mb region and whether differential DNA methylation of 2q14.2 genes could provide a regional panel of prostate cancer biomarkers., Methods: We used highly sensitive DNA methylation headloop PCR assays that can detect 10 to 25 pg of methylated DNA with a specificity of at least 1:1,000, and chromatin immunoprecipitation assays to investigate regional epigenetic remodeling across 2q14.2 in prostate cancer, in a cohort of 195 primary prostate tumors and 90 matched normal controls., Results: Prostate cancer cells exhibit concordant deacetylation and methylation of histone H3 Lysine 9 (H3K9Ac and H3K9me2, respectively), and localized DNA hypermethylation of EN1, SCTR, and INHBB and corresponding loss of H3K27me3. EN1 and SCTR were frequently methylated (65% and 53%, respectively), whereas INHBB was less frequently methylated., Conclusions: Consistent with LRES in colorectal cancer, we found regional epigenetic remodeling across 2q14.2 in prostate cancer. Concordant methylation of EN1 and SCTR was able to differentiate cancer from normal (P < 0.0001) and improved the diagnostic specificity of GSTP1 methylation for prostate cancer detection by 26%., Impact: For the first time we show that DNA methylation of EN1 and SCTR promoters provide potential novel biomarkers for prostate cancer detection and in combination with GSTP1 methylation can add increased specificity and sensitivity to improve diagnostic potential., (©2011 AACR.)

Background and Aims: Chronic pancreatitis and pancreatic cancer are characterised by extensive stellate cell mediated fibrosis, and current therapeutic development includes targeting pancreatic cancer stroma and tumor-host interactions. Recent evidence has suggested that circulating bone marrow derived stem cells (BMDC) contribute to solid organs. We aimed to define the role of circulating haematopoietic cells in the normal and diseased pancreas., Methods: Whole bone marrow was harvested from male β-actin-EGFP donor mice and transplanted into irradiated female recipient C57/BL6 mice. Chronic pancreatitis was induced with repeat injections of caerulein, while carcinogenesis was induced with an intrapancreatic injection of dimethylbenzanthracene (DMBA). Phenotype of engrafted donor-derived cells within the pancreas was assessed by immunohistochemistry, immunofluorescence and in situ hybridisation., Results: GFP positive cells were visible in the exocrine pancreatic epithelia from 3 months post transplantation. These exhibited acinar morphology and were positive for amylase and peanut agglutinin. Mice administered caerulein developed chronic pancreatitis while DMBA mice exhibited precursor lesions and pancreatic cancer. No acinar cells were identified to be donor-derived upon cessation of cerulein treatment, however rare occurrences of bone marrow-derived acinar cells were observed during pancreatic regeneration. Increased recruitment of BMDC was observed within the desmoplastic stroma, contributing to the activated pancreatic stellate cell (PaSC) population in both diseases. Expression of stellate cell markers CELSR3, PBX1 and GFAP was observed in BMD cancer-associated PaSCs, however cancer-associated, but not pancreatitis-associated BMD PaSCs, expressed the cancer PaSC specific marker CELSR3., Conclusions: This study demonstrates that BMDC can incorporate into the pancreas and adopt the differentiated state of the exocrine compartment. BMDC that contribute to the activated PaSC population in chronic pancreatitis and pancreatic cancer have different phenotypes, and may play important roles in these diseases. Further, bone marrow transplantation may provide a useful model for the study of tumor-host interactions in cancer and pancreatitis.

Purpose: Accurate estimates of recurrence risk are needed for optimal treatment of patients with clinically localized prostate cancer. We combined an established nomogram and what to our knowledge are novel molecular predictors into a new prognostic model of prostate specific antigen recurrence., Materials and Methods: We analyzed gene expression profiles from formalin fixed, paraffin embedded, localized prostate cancer tissues to identify genes associated with prostate specific antigen recurrence. Profiles of the identified markers were reproduced by reverse transcriptase-polymerase chain reaction. We used the profiles of 3 of these genes along with output from the Kattan postoperative nomogram to produce a predictive model of prostate specific antigen recurrence., Results: After variable selection we built a model of prostate specific antigen recurrence combining expression values of 3 genes and the postoperative nomogram. The 3-gene plus nomogram model predicted 5-year prostate specific antigen recurrence with a concordance index of 0.77 in a validation set compared to a concordance index of 0.67 for the nomogram. This model identified a subgroup of patients at high risk for recurrence that was not identified by the nomogram., Conclusions: This new gene based classifier has superior predictive power compared to that of the 5-year nomogram to assess the risk of prostate specific antigen recurrence in patients with organ confined prostate cancer. Our classifier should provide more accurate stratification of patients into high and low risk groups for treatment decisions and adjuvant clinical trials., (Copyright © 2010 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Background: Epigenetic alterations are common in prostate cancer, yet how these modifications contribute to carcinogenesis is poorly understood. We investigated whether specific histone modifications are prognostic for prostate cancer relapse, and whether the expression of epigenetic genes is altered in prostate tumorigenesis., Methods: Global levels of histone H3 lysine-18 acetylation (H3K18Ac) and histone H3 lysine-4 dimethylation (H3K4diMe) were assessed immunohistochemically in a prostate cancer cohort of 279 cases. Epigenetic gene expression was investigated in silico by analysis of microarray data from 23 primary prostate cancers (8 with biochemical recurrence and 15 without) and 7 metastatic lesions., Results: H3K18Ac and H3K4diMe are independent predictors of relapse-free survival, with high global levels associated with a 1.71-fold (P < 0.0001) and 1.80-fold (P = 0.006) increased risk of tumor recurrence, respectively. High levels of both histone modifications were associated with a 3-fold increased risk of relapse (P < 0.0001). Epigenetic gene expression profiling identified a candidate gene signature (DNMT3A, MBD4, MLL2, MLL3, NSD1, and SRCAP), which significantly discriminated nonmalignant from prostate tumor tissue (P = 0.0063) in an independent cohort., Conclusions: This study has established the importance of histone modifications in predicting prostate cancer relapse and has identified an epigenetic gene signature associated with prostate tumorigenesis., Impact: Our findings suggest that targeting the epigenetic enzymes specifically involved in a particular solid tumor may be a more effective approach. Moreover, testing for aberrant expression of epigenetic genes such as those identified in this study may be beneficial in predicting individual patient response to epigenetic therapies., (©2010 AACR.)

The phospholipid transfer protein STARD10 cooperates with c-erbB signaling and is overexpressed in Neu/ErbB2 breast cancers. We investigated if STARD10 expression provides additional prognostic information to HER2/neu status in primary breast cancer. A published gene expression dataset was used to determine relationships between STARD10 and HER2 mRNA levels and patient outcome. The central findings were independently validated by immunohistochemistry in a retrospective cohort of 222 patients with breast cancer with a median follow-up of 64 months. Kaplan-Meier and Cox proportional hazards analyses were used for univariate and multivariate analyses. Patients with low STARD10 or high HER2 tumor mRNA levels formed discrete groups each associated with a poor disease-specific survival (p = 0.0001 and p = 0.0058, respectively). In the immunohistochemical study low/absent STARD10 expression i.e. < or = 10% positive cells was observed in 24 of 222 (11%) tumors. In a univariate model, low/absent STARD10 expression was significantly associated with decreased patient survival (p = 0.0008). In multivariate analyses incorporating tumor size, tumor grade, lymph node status, ER, PR and HER2 status, low STARD10 expression was an independent predictor of death from breast cancer (HR: 2.56 (95% CI: 1.27-5.18), p = 0.0086). Furthermore, low/absent STARD10 expression, HER2 amplification and triple negative status were independent prognostic variables. Loss of STARD10 expression may provide an additional marker of poor outcome in breast cancer identifying a subgroup of patients with a particularly adverse prognosis, which is independent of HER2 amplification and the triple negative phenotype.

Objective: To investigate the effect of prostate-specific antigen (PSA) testing on stage migration in an Australian population, and its consequences on the prognostic accuracy of the post-radical prostatectomy (RP) Kattan nomogram, as in North America widespread PSA testing has resulted in prostate cancer stage migration, questioning the utility of prognostic nomograms in this setting., Patients and Methods: The study comprised 1008 men who had consecutive RP for localized prostate cancer between 1991 and 2001 at one institution. Two groups were assessed, i.e. those treated in 1991-96 (group 1, the early PSA era), and 1997-2001 (group 2, the contemporary PSA era). Differences in clinicopathological features between the groups were analysed by chi-squared testing and survival modelling. Individual patient data were entered into the post-RP Kattan nomogram and the efficacy assessed by receiver- operating characteristic curve analysis., Results: Patients in group 2 had lower pathological stage disease (P = 0.01) and fewer cancers with Gleason score > or =8 (P < 0.001) than group 1. Multivariate analysis identified preoperative serum PSA level (P < 0.01) and Gleason score (P < 0.01) as strong predictors of biochemical relapse in both groups. In group 2 pathological stage was not significant, but margin involvement became highly significant (P = 0.004). There was no difference in the predictive accuracy of the Kattan nomogram between the groups (P = 0.253)., Conclusions: These findings show a downward stage migration towards organ-confined disease after the introduction of widespread PSA testing in an Australian cohort. Despite this, the Kattan nomogram remains a robust prognostic tool in clinical practice.

GATA-2, a member of the GATA family of transcription factors, is involved in androgen receptor (AR) signaling, however, little is known regarding its role in prostate cancer. Here, we report that GATA-2 is expressed in a substantial proportion of prostate cancers and that high expression of GATA-2 is associated with biochemical recurrence and distant metastatic progression in a validation set of 203 cancers. In vitro data show that GATA-2 is directly recruited to the promoter region of the AR upon androgen stimulation of LNCaP prostate cancer cells with 5alpha-dihydroxytestosterone (DHT) for 24 h. Ectopic GATA-2 expression causes the induction of AR transcript levels under androgen-depleted conditions (P<0.05). The expression of the AR target gene, AZGP1, is induced upon androgen stimulation and this effect is repressed by GATA-2. In contrast, GATA-2 significantly increases transcript levels of KLK2, which increases further in a time-dependent manner on DHT treatment and in the presence of GATA-2. These results indicate that upregulation of GATA-2 may contribute to the progression to aggressive prostate cancer through modulation of expression of AR and key androgen-regulated genes, one of which, AZGP1, is associated with the progression to metastatic disease.

Docetaxel chemotherapy improves symptoms and survival in men with metastatic hormone-refractory prostate cancer (HRPC). However, approximately 50% of patients do not respond to Docetaxel and are exposed to significant toxicity without direct benefit. This study aimed to identify novel therapeutic targets and predictive biomarkers of Docetaxel resistance in HRPC. We used iTRAQ-mass spectrometry analysis to identify proteins associated with the development of Docetaxel resistance using Docetaxel-sensitive PC3 cells and Docetaxel-resistant PC3-Rx cells developed by Docetaxel dose escalation. Functional validation experiments were performed using recombinant protein treatment and siRNA knockdown experiments. Serum/plasma levels of the targets in patient samples were measured by ELISA. The IC(50) for Docetaxel in the PC3-Rx cells was 13-fold greater than the parent PC-3 cell line (P = 0.004). Protein profiling identified MIC-1 and AGR2 as respectively up-regulated and down-regulated in Docetaxel-resistant cells. PC-3 cells treated with recombinant MIC-1 also became resistant to Docetaxel (P = 0.03). Conversely, treating PC3-Rx cells with MIC-1 siRNA restored sensitivity to Docetaxel (P = 0.02). Knockdown of AGR2 expression in PC3 cells resulted in Docetaxel resistance (P = 0.007). Furthermore, increased serum/plasma levels of MIC-1 after cycle one of chemotherapy were associated with progression of the cancer (P = 0.006) and shorter survival after treatment (P = 0.002). These results suggest that both AGR2 and MIC-1 play a role in Docetaxel resistance in HRPC. In addition, an increase in serum/plasma MIC-1 level after cycle one of Docetaxel may be an indication to abandon further treatment. Further investigation of MIC-1 as a biomarker and therapeutic target for Docetaxel resistance in HRPC is warranted.

Purpose: Alternative CCND1 splicing results in cyclin D1b, which has specialized, protumorigenic functions in prostate not shared by the cyclin D1a (full length) isoform. Here, the frequency, tumor relevance, and mechanisms controlling cyclin D1b were challenged., Experimental Design: First, relative expression of both cyclin D1 isoforms was determined in prostate adenocarcinomas. Second, relevance of the androgen axis was determined. Third, minigenes were created to interrogate the role of the G/A870 polymorphism (within the splice site), and findings were validated in primary tissue. Fourth, the effect of G/A870 on cancer risk was assessed in two large case-control studies., Results: Cyclin D1b is induced in tumors, and a significant subset expressed this isoform in the absence of detectable cyclin D1a. Accordingly, the isoforms showed noncorrelated expression patterns, and hormone status did not alter splicing. Whereas G/A870 was not independently predictive of cancer risk, A870 predisposed for transcript-b production in cells and in normal prostate. The influence of A870 on overall transcript-b levels was relieved in tumors, indicating that aberrations in tumorigenesis likely alter the influence of the polymorphism., Conclusions: These studies reveal that cyclin D1b is specifically elevated in prostate tumorigenesis. Cyclin D1b expression patterns are distinct from that observed with cyclin D1a. The A870 allele predisposes for transcript-b production in a context-specific manner. Although A870 does not independently predict cancer risk, tumor cells can bypass the influence of the polymorphism. These findings have major implications for the analyses of D-cyclin function in the prostate and provide the foundation for future studies directed at identifying potential modifiers of the G/A870 polymorphism.

Background & Aims: Current methods of preoperative staging and predicting outcome following pancreatectomy for pancreatic cancer (PC) are inadequate. We evaluated the utility of multiple biomarkers from distinct biologic pathways as potential predictive markers of response to pancreatectomy and patient survival., Methods: We assessed the relationship of candidate biomarkers known, or suspected, to be aberrantly expressed in PC, with disease-specific survival and response to therapy in a cohort of 601 patients., Results: Of the 17 candidate biomarkers examined, only elevated expression of S100A2 was an independent predictor of survival in both the training (n = 162) and validation sets (n = 439; hazard ratio [HR], 2.19; 95% confidence interval [CI]: 1.48-3.25; P < .0001) when assessed in a multivariate model with clinical variables. Patients with high S100A2 expressing tumors had no survival benefit with pancreatectomy compared with those with locally advanced disease, whereas those without high S100A2 expression had a survival advantage of 10.6 months (19.4 vs 8.8 months, respectively) and a HR of 3.23 (95% CI: 2.39-4.33; P < .0001). Of significance, patients with S100A2-negative tumors had a significant survival benefit from pancreatectomy even in the presence of involved surgical margins (median, 15.7 months; P = .0007) or lymph node metastases (median, 17.4 months; P = .0002)., Conclusions: S100A2 expression is a good predictor of response to pancreatectomy for PC and suggests that high S100A2 expression may be a marker of a metastatic phenotype. Prospective measurement of S100A2 expression in diagnostic biopsy samples has potential clinical utility as a predictive marker of response to pancreatectomy and other therapies that target locoregional disease.

There is emerging evidence that the balance between estrogen receptor-alpha (ER(alpha)) and androgen receptor (AR) signaling is a critical determinant of growth in the normal and malignant breast. In this study, we assessed AR status in a cohort of 215 invasive ductal breast carcinomas. AR and (ER(alpha)) were coexpressed in the majority (80-90%) of breast tumor cells. Kaplan-Meier product limit analysis and multivariate Cox regression showed that AR is an independent prognostic factor in (ER(alpha))-positive disease, with a low level of AR (less than median of 75% positive cells) conferring a 4.6-fold increased risk of cancer-related death (P = 0.002). Consistent with a role for AR in breast cancer outcome, AR potently inhibited (ER(alpha))transactivation activity and 17beta-estradiol-stimulated growth of breast cancer cells. Transfection of MDA-MB-231 breast cancer cells with either functionally impaired AR variants or the DNA-binding domain of the AR indicated that the latter is both necessary and sufficient for inhibition of (ER(alpha)) signaling. Consistent with molecular modeling, electrophoretic mobility shift assays showed binding of the AR to an estrogen-responsive element (ERE). Evidence for a functional interaction of the AR with an ERE in vivo was provided by chromatin immunoprecipitation data, revealing recruitment of the AR to the progesterone receptor promoter in T-47D breast cancer cells. We conclude that, by binding to a subset of EREs, the AR can prevent activation of target genes that mediate the stimulatory effects of 17beta-estradiol on breast cancer cells.

Purpose: Current adjuvant therapies for pancreatic cancer (PC) are inconsistently used and only modestly effective. Because a high proportion of patients who undergo resection for PC likely harbor occult metastatic disease, any adjuvant trials assessing therapies such as radiotherapy directed at locoregional disease are significantly underpowered. Stratification based on the probability (and volume) of residual locoregional disease could play an important role in the design of future clinical trials assessing adjuvant radiotherapy., Patients and Methods: We assessed the relationships between margin involvement, the proximity to operative resection margins and outcome in a cohort of 365 patients who underwent operative resection for PC., Results: Microscopic involvement of a resection margin by tumor was associated with a poor prognosis. Stratifying the minimum clearance of resection margins by 0.5-mm increments demonstrated that although median survival was no different to clear margins based on these definitions, it was not until the resection margin was clear by more than 1.5 mm that optimal long-term survival was achieved., Conclusion: These data demonstrate that a margin clearance of more than 1.5 mm is important for long-term survival in a subgroup of patients. More aggressive therapeutic approaches that target locoregional disease such as radiotherapy may be beneficial in patients with close surgical margins. Stratification of patients for entry onto future clinical trials based on this criterion may identify those patients who benefit from adjuvant radiotherapy.

Aberrant expression of cyclin D1 protein is a common feature of breast cancer. However, the CCND1 gene encodes two gene products, cyclin D1a and cyclin D1b, which have discrete mechanisms of regulation and impact on cell behavior. A polymorphism at nucleotide 870 in the CCND1 gene, rs603965, influences the relative production of the encoded proteins and can impart increased risk for tumor development. Here, the impact of both the G/A870 polymorphism and cyclin D1b protein production on breast cancer risk, disease phenotype and patient outcome was analysed. In a large multiethnic case-control study, the G/A870 polymorphism conferred no significant risk for breast cancer overall or by stage or estrogen receptor (ER) status. However, the cyclin D1b protein was found to be upregulated in breast cancer, independent of cyclin D1a levels, and exhibited heterogeneous levels in breast cancer specimens. High cyclin D1a expression inversely correlated with the Ki67 proliferation marker and was not associated with clinical outcome. In contrast, elevated cyclin D1b expression was independently associated with adverse outcomes, including recurrence, distant metastasis and decreased survival. Interestingly, cyclin D1b was particularly associated with poor outcome in the context of ER-negative breast cancer. Thus, specific cyclin D1 isoforms are associated with discrete forms of breast cancer and high cyclin D1b protein levels hold prognostic potential.

Steroid hormones and their metabolising enzymes have been studied extensively for their potential role in prostate cancer, with more recent interest in the androgen/estrogen inactivating enzyme 17beta-hydroxysteroid dehydrogenase type 4 (HSD17B4). Gene expression profiling showed HSD17B4 to be significantly overexpressed in prostate cancer compared to matched-benign epithelium. We therefore hypothesized that altered HSD17B4 expression may contribute to prostate cancer progression via altered hormone balance. In this study, HSD17B4 mRNA and protein expression were assessed by in situ hybridisation (ISH) and immunohistochemistry (IHC), respectively, in tissue arrays of prostate tissue from 172 patients treated by radical prostatectomy. Overexpression of HSD17B4 mRNA and protein was associated with prostate cancer (P<0.0001) and multivariate Cox proportional hazards analysis, adjusted for known prognostic indicators, demonstrated HSD17B4 mRNA and high protein expression were significant independent predictors of poor patient outcome as measured by time until PSA relapse (mRNA: hazards ratio [HR]=1.90, 95% confidence interval [CI]=1.15-3.12; P<0.0001; and protein: HR=2.09, 95% CI=1.31-3.33; P=0.0026). Here we provide strong evidence that both mRNA and protein overexpression of HSD17B4 is not only associated with the presence of prostate cancer, but is also a significant independent predictor of poor patient outcome.

BAG-1 (bcl-2-associated athanogene) enhances oestrogen receptor (ER) function and may influence outcome and response to endocrine therapy in breast cancer. We determined relationships between BAG-1 expression, molecular phenotype, response to tamoxifen therapy and outcome in a cohort of breast cancer patients and its influence on tamoxifen sensitivity in MCF-7 breast cancer cells in vitro. Publically available gene expression data sets were analysed to identify relationships between BAG-1 mRNA expression and patient outcome. BAG-1 protein expression was assessed using immunohistochemistry in 292 patients with invasive ductal carcinoma and correlated with clinicopathological variables, therapeutic response and disease outcome. BAG-1-overexpressing MCF-7 cells were treated with antioestrogens to assess its effects on cell proliferation. Gene expression data demonstrated a consistent association between high BAG-1 mRNA and improved survival. In ER+ cancer (n=189), a high nuclear BAG-1 expression independently predicted improved outcome for local recurrence (P=0.0464), distant metastases (P=0.0435), death from breast cancer (P=0.009, hazards ratio 0.29, 95% CI: 0.114-0.735) and improved outcome in tamoxifen-treated patients (n=107; P=0.0191). BAG-1 overexpression in MCF-7 cells augmented antioestrogen-induced growth arrest. A high BAG-1 expression predicts improved patient outcome in ER+ breast carcinoma. This may reflect both a better definition of the hormone-responsive phenotype and a concurrent increased sensitivity to tamoxifen.

Background: Morphologically similar cancers display heterogeneous patterns of molecular aberrations and follow substantially different clinical courses. This diversity has become the basis for the definition of molecular phenotypes, with significant implications for therapy. Microarray or proteomic expression profiling is conventionally employed to identify disease-associated genes, however, traditional approaches for the analysis of profiling experiments may miss molecular aberrations which define biologically relevant subtypes., Methodology/principal Findings: Here we present Messina, a method that can identify those genes that only sometimes show aberrant expression in cancer. We demonstrate with simulated data that Messina is highly sensitive and specific when used to identify genes which are aberrantly expressed in only a proportion of cancers, and compare Messina to contemporary analysis techniques. We illustrate Messina by using it to detect the aberrant expression of a gene that may play an important role in pancreatic cancer., Conclusions/significance: Messina allows the detection of genes with profiles typical of markers of molecular subtype, and complements existing methods to assist the identification of such markers. Messina is applicable to any global expression profiling data, and to allow its easy application has been packaged into a freely-available stand-alone software package.

Background: MRP4/ABCC4 is an ATP-binding cassette transporter expressed in normal prostate. This study aimed to define the pattern of MRP4/ABCC4 expression in normal and malignant prostate tissue and the relationship of MRP4/ABCC4 expression and function in response to androgen signaling., Methods: Eighty-four radical prostatectomy specimens from patients with localized prostate cancer (PC) (22 neoadjuvant androgen ablation, AA, 62 no AA), 42 non-cancer and 16 advanced PCs were assessed for MRP4/ABCC4 mRNA/protein expression. The effect of DHT and bicalutamide on LNCaP cells was assessed by immunoblotting. HEK293 cells (+/-MRP4/ABCC4) were assessed for the ability to efflux androgens and anti-androgens., Results: MRP4/ABCC4 mRNA/protein levels were higher in localized PC compared to non-cancer (P = 0.006). MRP4/ABCC4 levels were significantly decreased in PCs treated with AA compared to cancers exposed to normal testosterone levels (P < 0.0001). MRP4/ABCC4 expression in normal human tissues was limited to the prostate and the renal tubules. MRP4/ABCC4 protein levels increased in LNCaP cells after DHT which was partially blocked by bicalutamide. However, DHT did not alter the activation of the MRP4/ABCC4 promotor in luciferase reporter assays and testosterone, DHT, flutamide and hydroxy-flutamide were not substrates for MRP4/ABCC4., Discussion: Elevated MRP4/ABCC4 expression is found in malignant compared to benign prostate tissue while lower MRP4/ABCC4 expression is seen after AA. Furthermore, MRP4/ABCC4 is upregulated by androgen and downregulated by anti-androgen treatment in vitro potentially through an indirect mode of action. These data strongly suggest that MRP4/ABCC4 is an androgen-regulated gene important in the progression to PC and may be a potential drug target., ((c) 2008 Wiley-Liss, Inc.)

The glycosaminoglycan chondroitin sulfate is significantly increased in the peritumoral stroma of prostate tumors compared with normal stroma and is an independent predictor of prostate-specific antigen (PSA) relapse following radical prostatectomy. In this study, we determined whether specific alterations in the sulfation pattern of glycosaminoglycan chains in clinically organ-confined prostate cancer are associated with PSA relapse. Immunoreactivity to distinct glycosaminoglycan disaccharide epitopes was assessed by manually scoring the staining intensity in prostate tissues from patients with benign prostatic hyperplasia (n = 19), early-stage cancer (cohort 1, n = 55 and cohort 2, n = 275), and advanced-stage cancer (n = 20). Alterations to glycosaminoglycans in benign and malignant prostate tissues were determined by cellulose acetate chromatography and high-pressure liquid chromatography. Glycosaminoglycan disaccharide epitopes were localized to the peritumoral stroma of clinically localized prostate cancer. The level of immunostaining for unsulfated disaccharides (C0S) in the peritumoral stroma, but not for 4-sulfated (C4S) or 6-sulfated disaccharides (C6S), was significantly associated with the rate of PSA relapse following radical prostatectomy. High levels of C0S immunostaining were determined to be an independent predictor of PSA relapse (1.6-fold, P = 0.020). Advanced-stage prostate cancer tissues exhibited reduced electrophoretic mobility for chondroitin sulfate and increased unsulfated disaccharides when compared with benign prostatic hyperplasia tissues, whereas the sulfated disaccharide levels were unaffected. The level of C0S immunostaining in the peritumoral stroma is an independent determinant of PSA failure in clinically localized prostate cancer. Specific alterations to chondroitin sulfate side chains occurring during tumor development may be a crucial step for disease progression in prostate cancer.

Cyclin D1 is a key mediator of cell cycle progression that is aberrantly regulated in multiple cancers, especially in breast cancers. A number of studies have indicated that a polymorphism in a splice donor site in the cyclin D1 gene is associated with alternative splicing and the production of the alternative cyclin D1b transcript. Furthermore, this polymorphism is selectively associated with disease outcomes. However, relatively little is known regarding the protein product of the alternatively spliced message, cyclin D1b. Using antibodies specific for cyclin D1b, it was found that this protein is readily detectable in a number of cancer cell lines and primary breast cancers. Whereas cyclin D1b interacts with cyclin-dependent kinase 4 (CDK4), it is relatively inefficient at mediating RB phosphorylation and cell cycle progression in model systems due to the lack of exon 5 of cyclin D1-encoded sequences. However, cyclin D1b protein levels are not significantly attenuated by DNA damage or antiestrogen treatment, indicating that the protein may have significant effect on the response to such therapeutic modalities. Whereas enforced expression of cyclin D1b was not sufficient to abrogate DNA damage checkpoint responses, it did efficiently overcome cell cycle arrest mediated by antiestrogen therapeutics. This action of cyclin D1b was not associated with effects on estrogen receptor activity, but was rather dependent on functional association with CDK4. Combined, these studies indicate that the cyclin D1b protein is aberrantly regulated and could contribute to therapeutic failure in the context of ER-positive breast cancer.

Despite a high initial response rate to first-line platinum/paclitaxel chemotherapy, most women with epithelial ovarian cancer relapse with recurrent disease that becomes refractory to further cytotoxic treatment. We have previously shown that the E3 ubiquitin ligase, EDD, a regulator of DNA damage responses, is amplified and overexpressed in serous ovarian carcinoma. Given that DNA damage pathways are linked to platinum resistance, the aim of this study was to determine if EDD expression was associated with disease recurrence and platinum sensitivity in serous ovarian cancer. High nuclear EDD expression, as determined by immunohistochemistry in a cohort of 151 women with serous ovarian carcinoma, was associated with an approximately two-fold increased risk of disease recurrence and death in patients who initially responded to first-line chemotherapy, independently of disease stage and suboptimal debulking. Although EDD expression was not directly correlated with relative cisplatin sensitivity of ovarian cancer cell lines, sensitivity to cisplatin was partially restored in platinum-resistant A2780-cp70 ovarian cancer cells following siRNA-mediated knockdown of EDD expression. These results identify EDD as a new independent prognostic marker for outcome in serous ovarian cancer, and suggest that pathways involving EDD, including DNA damage responses, may represent new therapeutic targets for chemoresistant ovarian cancer.

Identification of a biomarker of prognosis and response to therapy that can be assessed preoperatively would significantly improve overall outcomes for patients with pancreatic cancer. In this study, patients whose tumours exhibited high LMO4 expression had a significant survival advantage following operative resection, whereas the survival of those patients whose tumours had low or no LMO4 expression was not significantly different when resection was compared with operative biopsy alone.

Pancreatic ductal adenocarcinoma (PDA) is an extremely aggressive malignancy, which carries a dismal prognosis. Activating mutations of the Kras gene are common to the vast majority of human PDA. In addition, recent studies have demonstrated that embryonic signaling pathway such as Hedgehog and Notch are inappropriately upregulated in this disease. The role of another embryonic signaling pathway, namely the canonical Wnt cascade, is still controversial. Here, we use gene array analysis as a platform to demonstrate general activation of the canonical arm of the Wnt pathway in human PDA. Furthermore, we provide evidence for Wnt activation in mouse models of pancreatic cancer. Our results also indicate that Wnt signaling might be activated downstream of Hedgehog signaling, which is an early event in PDA evolution. Wnt inhibition blocked proliferation and induced apoptosis of cultured adenocarcinoma cells, thereby providing evidence to support the development of novel therapeutical strategies for Wnt inhibition in pancreatic adenocarcinoma.

Background: Secreted frizzled-related proteins (sFRP4) inhibits Wnt signaling and thus cellular proliferation in androgen-independent prostate cancer cells in vitro. However, increased expression of membranous sFRP4 is associated with a good prognosis in human localized androgen-dependent prostate cancer, suggesting a role for sFRP4 in early stage disease. Here, we investigated the phenotype of sFRP4 overexpression in an androgen-dependent prostate cancer model., Methods: An sFRP4-overexpressing androgen-dependent (LNCaP) prostate cancer model was established to assess changes in cellular proliferation, the expression, and subcellular localization of adhesion molecules and cellular invasiveness, and compared with the findings in sFRP4-overexpressing androgen-independent cells (PC3)., Results: sFRP4 overexpression in both cell line models resulted in a morphologic change to a more epithelioid cell type with increased localization of beta-catenin and cadherins (E-cadherin in LNCaP, N-cadherin in PC3) to the cell membrane. Functionally, sFRP4 overexpression was associated with a decreased rate of proliferation (P = 0.0005), decreased anchorage-independent growth (P < 0.001), and decreased invasiveness in PC3 cells (P < 0.0001). Furthermore, increased membranous sFRP4 expression was associated with increased membranous beta-catenin expression (P = 0.02) in a cohort of 224 localized human androgen-dependent prostate cancers., Conclusions: These data suggest that sFRP4 is an inhibitor of prostate cancer growth and invasion in vitro independent of androgen receptor (AR) signaling with correlative evidence in human androgen-dependent disease suggesting similar changes in the clinical setting. Consequently, potential therapeutic strategies to modulate Wnt signaling by sFRP4 will be relevant to both localized androgen-dependent prostate cancer and advanced metastatic disease.

The oncoprotein c-Myc is frequently overexpressed in breast cancer and ectopic expression in breast cancer cell lines attenuates responses to antiestrogen treatment. Here, we review preliminary data aimed at further elucidating a potential role for c-Myc in clinical endocrine resistance in breast cancer. Immunohistochemical and semi-quantitative PCR revealed that c-Myc protein and c-myc mRNA were frequently overexpressed in both ER-positive and ER-negative breast carcinoma. Furthermore, both constitutive and inducible c-Myc overexpression in MCF-7 breast cancer cell lines markedly reduced their sensitivity to the growth inhibitory effects of the pure antiestrogen ICI 182,780. In order to identify potential downstream targets of c-Myc that mediate this effect, Affymetrix microarrays were employed to examine the patterns of gene expression shared by MCF-7 cells stimulated by estrogen, or by induction of c-Myc. Approximately 50% of estrogen target genes identified 6h after treatment were also regulated by c-Myc. One novel target, EMU4, was transcriptionally regulated by c-Myc. In addition, there was a strong correlation between c-myc and EMU4 mRNA expression in a battery of breast cancer cell lines. These data confirm that c-Myc overexpression is a common event in breast cancer, and that this is associated with resistance to antiestrogens in vitro. Furthermore, the development of an experimental paradigm for the discovery of c-Myc and estrogen target genes associated with endocrine resistance provides a framework for the discovery and validation of genes involved in estrogen signalling, and c-Myc-mediated-antiestrogen resistance.

The risk of metastatic progression for prostate cancer patients who undergo radical prostatectomy is best estimated presently based on prostate-specific antigen (PSA) doubling time (PSADT). However, additional markers of risk are needed to identify patients who may benefit from aggressive salvage treatment. A decrease in zinc-alpha2-glycoprotein (AZGP1) mRNA levels in malignant prostate epithelium was previously shown to predict biochemical recurrence, as defined by rising levels of serum PSA after radical prostatectomy. We assessed the reliability with which AZPG1 expression could predict clinical recurrence and metastatic progression. Using immunohistochemical methods, we analyzed AZPG1 expression in malignant prostate epithelium in prostatectomy specimens from 228 prostate cancer patients. Low (i.e., absent or weak) AZGP1 expression was associated with clinical recurrence (defined as confirmed localized recurrence, metastasis, or death from prostate cancer; hazard ratio [HR] = 4.8, 95% confidence interval [CI] = 2.2 to 10.7, P<.001) and with bony metastases or death from prostate cancer (HR = 8.0, 95% CI = 2.6 to 24.3, P<.001). Among the 17 patients in the cohort in whom clinical recurrence was associated with short PSADT, absent or weak AZGP1 expression was observed in 13 patients. If these preliminary findings are validated in independent cohorts, the measurement of AZGP1 levels in radical prostatectomy specimens may permit an accurate and timely assessment of risk of metastatic progression after radical prostatectomy.

The association of high cyclin E expression with poor outcome in some cancers, in particular breast cancer, suggests that it may play an important role in tumor biology. Because the influence of cyclin E expression on outcome is yet to be examined in pancreatic cancer, we assessed the relationship between the expression of cyclin E, p27(Kip1), and survival in a large cohort of pancreatic cancer patients with long-term follow-up. Expression of cyclin E and p27(Kip1) was assessed by immunohistochemistry using tissue microarrays of tumor samples from 118 patients with pancreatic ductal adenocarcinoma (75 resections and 43 biopsies). High cyclin E expression (>10% positive nuclei) was identified in 39 of 118 (33%) patients. This was associated with poor prognosis on univariate analysis in the whole cohort (P = 0.005), as well as in the subgroup of 75 patients who underwent operative resection (P = 0.04). On multivariate analysis, high cyclin E expression was an independent predictor of poor survival in both the entire cohort (P = 0.005) and the resected subgroup (P = 0.03), and was superior to all tested clinicopathologic factors (tumor size, lymph node metastases, differentiation, margin involvement, and perineural invasion) as a marker of survival. Low p27(Kip1) expression (<5% positive nuclei) was present in 41 of 111 (37%) patients, but was not associated with survival, and coexpression of p27(Kip1) did not influence the association of high cyclin E expression with poor survival. High cyclin E expression is a strong independent predictor of poor outcome in patients with pancreatic cancer. Thus, if these data are confirmed in independent cohorts, measurement of cyclin E may add significant prognostic information to the currently used clinicopathologic variables and hence have potential clinical utility in the management of this disease.

Studies to elucidate dysregulated gene expression patterns in premalignant prostate lesions have identified several candidate genes with the potential to be targeted to prevent the development and progression of prostate cancer and act as biomarkers of early disease. Herein, we explored the importance of two proteins, neuropeptide Y (NPY) and macrophage inhibitory cytokine-1 (MIC-1), as biomarkers of preinvasive prostate disease and investigated the relationship of expression to biochemical recurrence following treatment for localized prostate cancer. NPY and MIC-1 protein expression was determined by immunohistochemistry on tissue microarrays containing 1,626 cores of benign, low-grade prostatic intraepithelial neoplasia (PIN), high-grade PIN (HGPIN), and prostate cancer tissue from 243 radical prostatectomy patients. Both NPY and MIC-1 showed higher proportional immunostaining in HGPIN and prostate cancer compared with benign epithelium (P < 0.0001). NPY and MIC-1 immunostaining was higher in low-grade PIN compared with other benign tissues (both P < 0.0001) and was equivalent to immunostaining in HGPIN. NPY immunostaining of prostate cancer was independently associated with relapse, after adjusting for traditional prognostic factors, as a categorical variable in 20% intervals (P = 0.0449-0.0103) and as a continuous variable (P = 0.0017). Low MIC-1 immunostaining (20% categories) was associated with pathologic stage >2C after adjusting for predictors of pathologic stage (P = 0.3894-0.0176). This is the first study to show that altered NPY and MIC-1 expression are significantly associated with prostate cancer progression and suggests that these molecules be developed further as biomarkers in the management of prostate disease.

Mucinous epithelial ovarian cancers (MOC) are clinically and morphologically distinct from the other histological subtypes of ovarian cancer. To determine the genetic basis of MOC and to identify potential tumour markers, gene expression profiling of 49 primary ovarian cancers of different histological subtypes was performed using a customised oligonucleotide microarray containing >59 000 probesets. The results show that MOC express a genetic profile that both differs and overlaps with other subtypes of epithelial ovarian cancer. Concordant with its histological phenotype, MOC express genes characteristic of mucinous carcinomas of varying epithelial origin, including intestinal carcinomas. Differences in gene expression between MOC and other histological subtypes of ovarian cancer were confirmed by RT-PCR and/or immunohistochemistry. In particular, galectin 4 (LGALS4) was highly and specifically expressed in MOC, but expressed at lower levels in benign mucinous cysts and borderline (atypical proliferative) tumours, supporting a malignant progression model of MOC. Hence LGALS4 may have application as an early and differential diagnostic marker of MOC.

Prostate cancer is the most common cancer in men. Advanced prostate cancer spreading beyond the gland is incurable. Identifying factors that regulate the spread of tumor into the regional nodes and distant sites would guide the development of novel diagnostic, prognostic, and therapeutic targets. The aim of our study was to examine the expression and biological role of EphB4 in prostate cancer. EphB4 mRNA is expressed in 64 of 72 (89%) prostate tumor tissues assessed. EphB4 protein expression is found in the majority (41 of 62, 66%) of tumors, and 3 of 20 (15%) normal prostate tissues. Little or no expression was observed in benign prostate epithelial cell line, but EphB4 was expressed in all prostate cancer cell lines to varying degrees. EphB4 protein levels are high in the PC3 prostate cancer cell line and several folds higher in a metastatic clone of PC3 (PC3M) where overexpression was accompanied by EphB4 gene amplification. EphB4 expression is induced by loss of PTEN, p53, and induced by epidermal growth factor/epidermal growth factor receptor and insulin-like growth factor-I/insulin-like growth factor-IR. Knockdown of the EphB4 protein using EphB4 short interfering RNA or antisense oligodeoxynucleotide significantly inhibits cell growth/viability, migration, and invasion, and induces apoptosis in prostate cancer cell lines. Antisense oligodeoxynucleotide targeting EphB4 in vivo showed antitumor activity in murine human tumor xenograft model. These data show a role for EphB4 in prostate cancer and provide a rationale to study EphB4 for diagnostic, prognostic, and therapeutic applications.

Gene expression profiling offers a promising new technique for the diagnosis and prognosis of cancer. We have applied this technology to build a clinically robust site of origin classifier with the ultimate aim of applying it to determine the origin of cancer of unknown primary (CUP). A single cDNA microarray platform was used to profile 229 primary and metastatic tumors representing 14 tumor types and multiple histologic subtypes. This data set was subsequently used for training and validation of a support vector machine (SVM) classifier, demonstrating 89% accuracy using a 13-class model. Further, we show the translation of a five-class classifier to a quantitative PCR-based platform. Selecting 79 optimal gene markers, we generated a quantitative-PCR low-density array, allowing the assay of both fresh-frozen and formalin-fixed paraffin-embedded (FFPE) tissue. Data generated using both quantitative PCR and microarray were subsequently used to train and validate a cross-platform SVM model with high prediction accuracy. Finally, we applied our SVM classifiers to 13 cases of CUP. We show that the microarray SVM classifier was capable of making high confidence predictions in 11 of 13 cases. These predictions were supported by comprehensive review of the patients' clinical histories.

Purpose: Despite significant progress in understanding the molecular pathology of pancreatic cancer and its precursor lesion: pancreatic intraepithelial neoplasia (PanIN), there remain no molecules with proven clinical utility as prognostic or therapeutic markers. Here, we used oligonucleotide microarrays to interrogate mRNA expression of pancreatic cancer tissue and normal pancreas to identify novel molecular pathways dysregulated in the development and progression of pancreatic cancer., Experimental Design: RNA was hybridized to Affymetrix Genechip HG-U133 oligonucleotide microarrays. A relational database integrating data from publicly available resources was created to identify candidate genes potentially relevant to pancreatic cancer. The protein expression of one candidate, homeobox B2 (HOXB2), in PanIN and pancreatic cancer was assessed using immunohistochemistry., Results: We identified aberrant expression of several components of the retinoic acid (RA) signaling pathway (RARalpha, MUC4, Id-1, MMP9, uPAR, HB-EGF, HOXB6, and HOXB2), many of which are known to be aberrantly expressed in pancreatic cancer and PanIN. HOXB2, a downstream target of RA, was up-regulated 6.7-fold in pancreatic cancer compared with normal pancreas. Immunohistochemistry revealed ectopic expression of HOXB2 in 15% of early PanIN lesions and 48 of 128 (38%) pancreatic cancer specimens. Expression of HOXB2 was associated with nonresectable tumors and was an independent predictor of poor survival in resected tumors., Conclusions: We identified aberrant expression of RA signaling components in pancreatic cancer, including HOXB2, which was expressed in a proportion of PanIN lesions. Ectopic expression of HOXB2 was associated with a poor prognosis for all patients with pancreatic cancer and was an independent predictor of survival in patients who underwent resection.

Molecular markers have the potential to serve not only as prognostic factors but may be targets for new therapeutic strategies and predictors of response in a range of cancers. Prostate cancer development and progression is predicated on a series of genetic and epigenetic events within the prostate cell and its milieu. Within this review, we identify candidate molecules involved in diverse processes such as cell proliferation, death and apoptosis, signal transduction, androgen receptor (AR) signalling, cellular adhesion and angiogenesis that are linked to outcome in prostate cancer. Current markers with potential prognostic value include p53, Bcl-2, p16INK4A, p27Kip1, c-Myc, AR, E-cadherin and vascular endothelial growth factor. Evolving technology permits the identification of an increasing number of molecular markers with prognosis and predictive potential. We also review the use of gene microarray analysis in gene discovery as a means of identifying and cosegregating novel markers of prostate cancer outcome. By integrating selected markers into prospective clinical trials, there is potential for us to provide specific targeted therapy tailored for an increasing number of patients.

The extracellular matrix (ECM) is a reservoir of cellular binding proteins and growth factors that are critical for normal cell behavior, and aberrations in the ECM invariably accompany malignancies such as prostate cancer. Carcinomas commonly overexpress macrophage inhibitory cytokine 1 (MIC-1), a proapoptotic and antitumorigenic transforming growth factor-beta superfamily cytokine. Here we show that MIC-1 is often secreted in an unprocessed propeptide containing form. It is variably processed intracellularly, with unprocessed forms being secreted from several tumor lines, including prostate carcinoma lines, PC-3 and LNCaP. Once secreted, only unprocessed proMIC-1 binds ECM, demonstrating for the first time the occurrence of extracellular stores of MIC-1. The propeptide mediates this association via its COOH-terminal 89 amino acids. Xenograft models bearing tumors secreting various engineered forms of MIC-1 show that the propeptide regulates the balance between ECM stores and circulating serum levels of mature MIC-1 in vivo. The absence of propeptide results in approximately 20-fold increase in serum MIC-1 levels. The significance of stromal MIC-1 stores was evaluated in prostate cancer tissue cores, which show major variation in stromal levels of MIC-1. Stromal MIC-1 levels are linked to prostate cancer outcome following radical prostatectomy, with decreasing stromal levels providing an important independent predictor of disease relapse. In low-grade localized prostate cancer (Gleason sum score < or = 6), the level of MIC-1 stromal stores was the best predictor of future relapse when compared with all other clinicopathologic variables. The secretion and ECM association of unprocessed proMIC-1 is likely to play a central role in modulating local bioavailability of MIC-1 which can affect patient outcome in prostate cancer and other epithelial tumors.

Beta-catenin in its role as a nuclear signaling molecule has been implicated in prostate carcinogenesis primarily through modulation of androgen receptor activity. We defined the pattern of beta-catenin protein expression in the nuclei of normal, hyperplastic and malignant human prostate tissue and determined whether differences in expression were associated with disease progression and prognosis. Five normal prostates, 26 benign prostatic hypertrophy specimens, 232 radical prostatectomy specimens from patients with clinically localized prostate cancer (PC) and 20 cases of advanced PC were assessed for beta-catenin expression using immunohistochemistry. Nuclear beta-catenin expression in localized PC was significantly lower than that in benign prostate tissue (p < 0.001) and significantly higher than that in advanced PC tissue (p < 0.001). In addition, lower levels of nuclear beta-catenin expression (< 10% of cancer cells) predicted for a shorter biochemical relapse-free survival in patients with localized PC (p = 0.008) and were inversely correlated with preoperative prostate-specific antigen (PSA) levels (p = 0.01). Analysis of the low-risk subgroup of patients with preoperative PSA levels < 10 ng/ml demonstrated that lower levels of nuclear beta-catenin expression (< 10% of cancer cells) again predicted for a poorer prognosis (p = 0.006). In conclusion, lower levels of nuclear beta-catenin expression are found in malignant compared to benign prostate tissue. In addition, lower nuclear beta-catenin expression is associated with a poorer prognosis in localized PC, in particular, in the low-risk subgroup of patients with preoperative PSA levels < 10 ng/ml. Thus, the level of nuclear beta-catenin expression may be of clinical utility as a preoperative prognostic marker in low-risk localized PC.