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NCOR1 signature

SIAH2 signature in LNCaP cells

Purpose: To assess the safety of administering bortezomib to patients undergoing a radical prostatectomy, to assess pathologic changes induced by bortezomib in prostate cancer specimen, and to verify alterations by the drug in proteasome protein targets.Experimental Design: Bortezomib is a proteasome inhibitor that has shown activity in vitro and in vivo in prostate cancer. We performed a neoadjuvant clinical trial of bortezomib in men with prostate cancer at high risk of recurrence. The primary endpoints were to evaluate safety and biological activity.Results: Bortezomib is generally safe in the preoperative setting. Antitumor activity was manifested by tumor cytopathic effect, drops in serum prostate-specific antigen in some patients, and increases in tumor apoptosis. This was associated with cytoplasmic entrapment of nuclear factor-κB. We found an unexpected increase in proliferation in treated tissues and in vitro. Bortezomib also increased SRC-3 levels and phosphorylated Akt, both in vitro and in treated prostate cancer tissues. Knockdown of SRC-3 blocked the increase in activated Akt in vitro. Combined treatment with bortezomib and the Akt inhibitor perifosine was more effective than either agent alone in vitro.Conclusion: These data suggest that combined therapies targeting the proteasome and the Akt pathway may have increased efficacy.

Sequences of siRNAs and primers and probes for qPCR

Correlation analysis between NCOR1 and NCOR2 expression and AR transcriptional output

Purpose: Castration therapy in advanced prostate cancer eventually fails and leads to the development of castration-resistant prostate cancer (CRPC), which has no cure. Characteristic features of CRPC can be increased androgen receptor (AR) expression and altered transcriptional output. We investigated the expression of nuclear receptor corepressor 1 (NCOR1) in human prostate and prostate cancer and the role of NCOR1 in response to antiandrogens.Experimental Design: NCOR1 protein levels were compared between matched normal prostate and prostate cancer in 409 patient samples. NCOR1 knockdown was used to investigate its effect on bicalutamide response in androgen-dependent prostate cancer cell lines and transcriptional changes associated with the loss of NCOR1. NCOR1 transcriptional signature was also examined in prostate cancer gene expression datasets.Results: NCOR1 protein was detected in cytoplasm and nuclei of secretory epithelial cells in normal prostate. Both cytoplasmic and nuclear NCOR1 protein levels were lower in prostate cancer than in normal prostate. Prostate cancer metastases show significant decrease in NCOR1 transcriptional output. Inhibition of LNCaP cellular proliferation by bicalutamide requires NCOR1. NCOR1-regulated genes suppress cellular proliferation and mediate bicalutamide resistance. In the mouse, NCOR1 is required for bicalutamide-dependent regulation of a subset of the AR target genes.Conclusions: In summary, we demonstrated that NCOR1 function declines with prostate cancer progression. Reduction in NCOR1 levels causes bicalutamide resistance in LNCaP cells and compromises response to bicalutamide in mouse prostate in vivo. Clin Cancer Res; 22(15); 3937–49. ©2016 AACR.

DHT regulated pathways

NCOR1 regulated pathways

AR signature in LNCaP cells

Supplementary Data from Bortezomib-Mediated Inhibition of Steroid Receptor Coactivator-3 Degradation Leads to Activated Akt

Supplementary Figure 1 Comparison of NCOR1 and NCOR2 knockdown in LAPC4 and LNCaP cells Supplementary Figure 2 NCOR1 regulates motility of LNCaP cells Supplementary Figure 3 AR signature is present among bicalutamide treated genes in both control and NCOR1 depleted cells Supplementary Figure 4 Comparative levels of UGT2B15 and UGT2B17 mRNA expression in LNCaP and C4-2 cells Supplementary Figure 5 Loss of both UGT2B 15 and UGT2B 17 lead to elevated DHT-dependent PSA expression. Supplementary Figure 6 NCOR1 is required for optimal TARP and CHEK1 expression in LAPC4 cells Supplementary Figure 7 AR is recruited to PSA enhancer and INPP4B promoter after NCOR1 depletion Supplementary Figure 8 Examples of variable NCOR1 staining in normal prostate epithelium, PIN, and prostate cancer on tissue microarray.

Supplementary Figure 1 from Androgens Modulate Expression of Transcription Intermediary Factor 2, an Androgen Receptor Coactivator whose Expression Level Correlates with Early Biochemical Recurrence in Prostate Cancer

Background: A number of studies have reported elevated incidence of 25-OH-vitamin D deficiency among patients with multiple myeloma (MM). Several studies have found association between vitamin D levels and factors associated with survival, including ISS stage at diagnosis. However, the impact of vitamin D deficiency on MM prognosis is not entirely clear. Also, in general, both the incidence and the impact of vitamin D deficiency differ substantially by race. Here, we investigate the impact of vitamin D deficiency on prognosis in a large and racially heterogenous patient population with MM in the Veterans Affairs (VA) system. Methods: We used the VA's nationwide Corporate Data Warehouse to identify patients diagnosed with symptomatic MM from 1999 to 2017. Various demographic and laboratory data was collected including age, race, 25-OH-vitamin D levels, and ISS stage at diagnosis as well as survival outcome data. Details of therapies received was also available which indicted similar access to all newer agents approved for myeloma for both African American (AA) and Caucasian patients. Results: We identified 15,717 patients diagnosed with MM (3353 AA and 9070 Caucasian), of whom 6675 had vitamin D measurements within 2 months of diagnosis (1959 AA and 4398 Caucasian). Median serum vitamin D levels were significantly lower among AA patients (21.8 ng/mL) than Caucasians (28.6 ng/mL; p We evaluated the ability of serum vitamin D level to predict overall survival (OS) in patients with MM using a cut-off of 20ng/mL. Patients with vitamin D deficiency ( The analyses were repeated for AA and Caucasian patients separately. Among AA patients, serum vitamin D was not a significant predictor of OS in univariate (P=0.5096) or multivariate analysis (P=0.6923), while it was still a strong predictor among Caucasian patients in both univariate (HR 1.38; P=0.0006) and multivariate analysis (HR 1.45; P=0.0048). Median OS is 3.54 years (95% CI 2.99-5.52; n=255) for AA patients with vitamin D deficiency and 3.95 years (3.25-5.35; n=296) with normal levels. Among Caucasians, median OS is 2.71 years (2.18-3.47; n=273) for deficient and 3.87 years (3.59-4.42; n=885) for normal. Kaplan-Meier plots (Fig. 1B and 1C) illustrate the observed OS curves for the two subgroups. Since levels of vitamin D were lower in AA patients, a lower cut-off of 10 ng/mL was also tested. Even using this lower cutoff, vitamin D deficiency was not a statistically significant predictor of OS in univariate (HR 1.33; P=0.0781) or multivariate analysis (HR 1.09; P=0.7039), though the number of AA patients with vitamin D Conclusions: Vitamin D deficiency is a significant predictor of survival among patients diagnosed with MM, even after accounting for race, age, and ISS stage. However, this relationship is only observed in Caucasian patients and not observed among AA patients. Studies are ongoing to evaluate impact of Vitamin D deficiency of disease presentation including bone disease as well as genetic characteristics. This investigation highlights the need to assess the underlying biological mechanism responsible for the observed impact of vitamin D deficiency across race in MM. Download : Download high-res image (145KB) Download : Download full-size image Disclosures Yellapragada: Novartis: Employment; Celgene: Research Funding; Takeda: Research Funding. Munshi: OncoPep: Other: Board of director.

Background Nerves are key factors in prostate cancer (PCa), but the functional role of innervation in prostate cancer is poorly understood. PCa induced neurogenesis and perineural invasion (PNI), are associated with aggressive disease. Method We denervated rodent prostates chemically and physically, before orthotopically implanting cancer cells. We also performed a human neoadjuvant clinical trial using botulinum toxin type A (Botox) and saline in the same patient, before prostatectomy. Result Bilateral denervation resulted in reduced tumor incidence and size in mice. Botox treatment in humans resulted in increased apoptosis of cancer cells in the Botox treated side. A similar denervation gene array profile was identified in tumors arising in denervated rodent prostates, in spinal cord injury patients and in the Botox treated side of patients. Denervation induced exhibited a signature gene profile, indicating translation and bioenergetic shutdown. Nerves also regulate basic cellular functions of non-neoplastic epithelial cells. Conclusion Nerves play a role in the homeostasis of normal epithelial tissues and are involved in prostate cancer tumor survival. This study confirms that interactions between human cancer and nerves are essential to disease progression. This work may make a major impact in general cancer treatment strategies, as nerve/cancer interactions are likely important in other cancers as well. Targeting the neural microenvironment may represent a therapeutic approach for the treatment of human prostate cancer.

Obesity is a common comorbidity of chronic obstructive pulmonary disease (COPD) and has been associated with worse outcomes. However, it is unknown whether the interaction between obesity and COPD modulates diaphragm shape and consequently its function. The body mass index (BMI) has been used as a correlate of obesity. We tested the hypothesis that the shape of the diaphragm muscle and size of the ring of its insertion in non-COPD and COPD subjects are modulated by BMI. We recruited 48 COPD patients with postbronchiodilator forced expiratory volume in 1 s (FEV1)-to-forced vital capacity (FVC) < 0.7 and 29 age-matched smoker/exsmoker control (non-COPD) subjects, who underwent chest computed tomography (CT) at lung volumes ranging from functional residual capacity (FRC) to total lung capacity (TLC). We then computed maximum principal diaphragm curvature in the midcostal region of the left hemidiaphragm at the end of inspiration during quiet breathing (EI) and at TLC. The radius of maximum curvature of diaphragm muscle increased with BMI in both COPD and non-COPD subjects. The size of diaphragm ring of insertion on the chest wall also increased significantly with increasing BMI. Surprisingly, COPD severity did not appear to cause significant alteration in diaphragm shape except in normal-weight subjects at TLC. Our data uncovered important factors such as BMI, the size of the diaphragm ring of insertion, and disease severity that modulate the structure of the ventilatory pump in non-COPD and COPD subjects.

Phospho-Akt (P-Akt1) promotes proliferation and increased survival in vitro and plays an important role in prostate cancer (PCa) progression as well as the prediction of the probability of recurrence. In this study, the goal was to demonstrate the involvement and impact of P-Akt1 on cellular interactions, biomechanisms, and pathways in PCa. Tissue microarrays from 640 PCa patients were immunostained with various antibodies. Ki-67 was used to measure proliferation index, and terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling was used for apoptotic index. Increased expression of P-Akt1 was associated with an increased proliferation but inversely correlated with apoptotic index. Higher levels of P-Akt1 are associated with both higher levels of cytoplasmic p27 and higher levels of nuclear p27, suggesting an involvement in both cytoplasmic entrapment and phosphorylation of p27. P-Akt1 expression significantly correlated with nuclear and cytoplasmic staining of FHKR and GSK. The strongest correlations were found with the P- forms of both, suggesting enzyme kinetics in the latter. Here, phosphorylation is the principal method of FHKR and GSK inactivation. P-Akt1 correlated with nuclear transcription factor kappa B, suggesting a role in the inhibition through phosphorylation of nuclear transcription factor kappa B. The results of the current study are unique because of the scope of the markers and the size of the population used. In vitro- and in vivo-derived information of P-Akt1 and its downstream effectors demonstrates significant involvement in PCa. Our data suggest that PCa uses multiple mechanisms to regulate this pathway and substantiate the concept of redundancy in cancer pathway regulation. Consequently, new hypothesis-driven studies can be derived from this information.

BACKGROUND TMPRSS2-ERG fusion occurs in about half of prostate cancers and results in over-expression of the oncogenic ERG protein in the prostate. The mechanism by which ERG contributes to prostate cancer initiation and progression remains largely unknown. Because ERG is a transcriptional activator, we reasoned that the target genes regulated by ERG could contribute to prostate cancer development. METHODS In a search for ERG target genes, we took advantage of published datasets from the MSKCC Prostate Oncogene Project, in which a comprehensive analysis was applied to define transcriptomes in 150 prostate tumors. We retrieved the mRNA expression dataset, split them based on ERG expression, and identified genes whose expression levels are associated with ERG mRNA levels. RESULTS mRNA expression levels of 21 genes were found to be significantly increased, while for one gene it was decreased in ERG-positive prostate tumors. Among them, the expression of TDRD1 was the most significantly increased in ERG-positive tumors. Among 131 primary prostate tumors which were primarily from European American patients, TDRD1 is over-expressed in 68% of samples, while ERG is overexpressed in 48% of samples, suggesting an additional ERG-independent mechanism of TDRD1 overexpression. In African American prostate tumors, TDRD1 mRNA is expressed in 44%, while ERG is expressed in 24% of samples. In normal tissues, TDRD1 mRNA is exclusively expressed in germ cells and its protein is also known as cancer/testis antigen 41.1 (CT41.1). We generated a mouse monoclonal antibody that recognizes human TDRD1 protein with high specificity and sensitivity. By Western blot analysis and immunohistochemistry (IHC) staining, we demonstrate that TDRD1 protein is expressed in the majority of human prostate tumors, but not in normal prostate tissue. Finally, TDRD1 is not induced in the prostate of ERG overexpression transgenic mice, suggesting that such model does not fully recapitulate the TMPRSS2/ERG fusion-dependent human prostate cancer development. CONCLUSIONS Our results suggest TDRD1 as a novel prostate cancer biomarker. As an ERG target gene, TDRD1 might play an important role in human prostate cancer development, and as a cancer/testis antigen, TDRD1 might have long-term potential to be a therapeutic target for prostate cancer immunotherapy. Prostate © 2016 Wiley Periodicals, Inc.

Prostate cancer is the second leading cause of cancer-related death of men in the USA. TheTMPRSS2/ERG (T/E)fusion gene is present in approximately 50% of prostate cancers and promotes tumor progressionin vivo. The presence of theT/Efusion gene is strongly associated with the expression of ERG protein, but emerging evidence indicates a significant interfocal and intrafocal variability in the levels of ERG protein expression. We therefore analyzed ERG protein expression by image analysis to objectively quantitate the extent of such heterogeneity, and confirmed significant interfocal and intrafocal variability of ERG protein expression levels in cancer expressing ERG. To define the pathways associated with ERG and its variable expression in prostate cancer, we have analyzed the correlations of ERG expression, as evaluated by immunohistochemistry, with 46 key proteins associated with signal transduction, transcriptional control, and other processes using a large tissue microarray with more than 500 prostate cancers. We found a significant correlation of ERG expression with the markers of activation of the PI3K, MYC, and NFκB pathways, which had previously been linked directly or indirectly to ERG expression. We have also identified significant correlations with novel proteins that have not been previously linked to ERG expression, including serum response factor, the p160 coactivator SRC1, and Sprouty1. Notably, SKP2 only correlated with a high level of ERG protein expression. Thus ERG expression is variable in prostate cancer and is associated with activation of multiple pathways and proteins including several potentially targetable pathways.

Background: Semaphorin 4F (S4F) has roles in embryologic axon guidance and is expressed in adults. S4F is involved in cancer-induced neurogenesis. Methods: Prostate cells were transfected with S4F retrovirus. Cells and controls were used for a bromodeoxyuridine (BrdUrd) incorporation assay (proliferation) and in vitro scratch and Matrigel Transwell chamber invasion assay (migration). Monoclonal antibodies were developed using baculovirus-expressed recombinant GST-S4F and used to immunostain tissue microarrays. Slides were imaged using deconvolution and analyzed using tissue segmentation. Data were correlated with clinicopathologic parameters, other biomarkers and survival analysis conducted. Heterogeneity of S4F expression was analyzed with unsupervised clustering algorithms. Results: Proliferation rates measured by BrdUrd incorporation were higher in all S4F-transfected cells. S4F overexpression was associated with increased motility of the cancer cells. S4F expression was overexpressed in high-grade prostatic intraepithelial neoplasia/prostate cancer than normal epithelium. S4F expression correlated with seminal vesicle invasion. Patients with high values of S4F in prostate cancer cytoplasm are at significantly higher risk of biochemical recurrence, by univariate and multivariate analyses. S4F cytoplasmic expression in prostate cancer cells also correlates with nerve density in prostate cancer and perineural invasion diameter. Correlations were identified with NF-κB and inversely with apoptosis in perineural invasion. Conclusion: These data show that S4F is significantly involved in human prostate cancer progression. S4F is a key regulator of the interactions between nerves in the tumor microenvironment and cancer cells. Because of the importance of cancer nerve interaction in the biology of cancer and its clinical implication, S4F can be considered a major therapeutic target. Clin Cancer Res; 19(22); 6101–11. ©2013 AACR.

Levels of caveolin-1 (Cav-1) in tumour epithelial cells increase during prostate cancer progression. Conversely, Cav-1 expression in the stroma can decline in advanced and metastatic prostate cancer. In a large cohort of 724 prostate cancers, we observed significantly decreased levels of stromal Cav-1 in concordance with increased Gleason score (p = 0.012). Importantly, reduced expression of Cav-1 in the stroma correlated with reduced relapse-free survival (p = 0.009), suggesting a role for stromal Cav-1 in inhibiting advanced disease. Silencing of Cav-1 by shRNA in WPMY-1 prostate fibroblasts resulted in up-regulation of Akt phosphorylation, and significantly altered expression of genes involved in angiogenesis, invasion, and metastasis, including a > 2.5-fold increase in TGF-β1 and γ-synuclein (SNCG) gene expression. Moreover, silencing of Cav-1 induced migration of prostate cancer cells when stromal cells were used as attractants. Pharmacological inhibition of Akt caused down-regulation of TGF-β1 and SNCG, suggesting that loss of Cav-1 in the stroma can influence Akt-mediated signalling in the tumour microenvironment. Cav-1-depleted stromal cells exhibited increased levels of intracellular cholesterol, a precursor for androgen biosynthesis, steroidogenic enzymes, and testosterone. These findings suggest that loss of Cav-1 in the tumour microenvironment contributes to the metastatic behaviour of tumour cells by a mechanism that involves up-regulation of TGF-β1 and SNCG through Akt activation. They also suggest that intracrine production of androgens, a process relevant to castration resistance, may occur in the stroma.

Mutations in phosphatase and tensin homologue (PTEN) or genomic alterations in the phosphatidylinositol-3-OH kinase-signalling pathway are the most common genetic alterations reported in human prostate cancer. However, the precise mechanism underlying how indolent tumours with PTEN alterations acquire metastatic potential remains poorly understood. Recent studies suggest that upregulation of transforming growth factor (TGF)-β signalling triggered by PTEN loss will form a growth barrier as a defence mechanism to constrain prostate cancer progression, underscoring that TGF-β signalling might represent a pre-invasive checkpoint to prevent PTEN-mediated prostate tumorigenesis. Here we show that COUP transcription factor II (COUP-TFII, also known as NR2F2), a member of the nuclear receptor superfamily, serves as a key regulator to inhibit SMAD4-dependent transcription, and consequently overrides the TGF-β-dependent checkpoint for PTEN-null indolent tumours. Overexpression of COUP-TFII in the mouse prostate epithelium cooperates with PTEN deletion to augment malignant progression and produce an aggressive metastasis-prone tumour. The functional counteraction between COUP-TFII and SMAD4 is reinforced by genetically engineered mouse models in which conditional loss of SMAD4 diminishes the inhibitory effects elicited by COUP-TFII ablation. The biological significance of COUP-TFII in prostate carcinogenesis is substantiated by patient sample analysis, in which COUP-TFII expression or activity is tightly correlated with tumour recurrence and disease progression, whereas it is inversely associated with TGF-β signalling. These findings reveal that the destruction of the TGF-β-dependent barrier by COUP-TFII is crucial for the progression of PTEN-mutant prostate cancer into a life-threatening disease, and supports COUP-TFII as a potential drug target for the intervention of metastatic human prostate cancer.

Purpose: Increased expression of FGFR-4 and its ligands have been linked to lethal prostate cancer (PCa). Furthermore, a germ line polymorphism in the FGFR-4 gene, resulting in arginine at codon 388 (Arg388) instead of glycine (Gly388), is associated with aggressive disease. The FGFR-4 Arg388 variant results in increased receptor stability, sustained receptor activation, and increased motility and invasion compared with Gly388. However, the impact of sustained signaling on cellular signal transduction pathways is unknown. Experimental Design: Expression microarray analysis of immortalized prostatic epithelial cells lines expressing FGFR-4 Arg388 or Gly388 was used to establish a gene signature associated with FGFR-4 Arg388 expression. Transient transfection of reporters and inhibitors was used to establish the pathways activated by FGFR-4 Arg388 expression. The impact of pathway knockdown in vitro and in an orthotopic model was assessed using inhibitors and/or short hairpin RNA (shRNA). Results: Expression of the FGFR-4 Arg388 protein leads to increased activity of the extracellular signal–related kinase (ERK) pathway, increased activity of serum response factor (SRF) and AP1, and transcription of multiple genes that are correlated with aggressive clinical behavior in PCa. Increased expression of SRF is associated with biochemical recurrence in men undergoing radical prostatectomy. Consistent with these observations, knockdown of FGFR-4 Arg388 in PCa cells decreases proliferation and invasion in vitro and primary tumor growth and metastasis in vivo. Conclusions: These studies define a signal transduction pathway downstream of FGFR-4 Arg388 that acts via ERK and SRF to promote PCa progression. Clin Cancer Res; 17(13); 4355–66. ©2011 AACR.

Introduction: Myelodysplastic syndrome (MDS) is a heterogeneous group of myeloid malignant disorders characterized by defects in hematopoietic progenitor differentiation and propensity for acute myelogenous leukemia (AML). Detectable DNMT3A, TET2, and ASXL1 mutations are not only associated with increased malignant conversion risk, but also with broad Cardiovascular (CV) risk. Indeed, clonal hematopoiesis of indetetermined potential (CHIP), is associated with parametric erythroid modifications (i.e., red cell distribution width [RDW] and mean corpuscular volume [MCV]) that occurs years before hematologic malignancy development, and correlates with current patient (pt) risk for CV disease. In this study, we seek to examine the predictive value of MCV in MDS pt diagnosed with and without CV disease. Methods: After IRB approval, 84 pt diagnosed with low-risk MDS (LR-MDS) were included for analysis. Median MCV was estimated in all LR-MDS patients to: (1) Examine overall survival (OS) in LR-MDS with and without CV disease; and (2) Detect differential OS in LR-MDS pt with and without CV disease according to baseline predictors such as hemoglobin, platelet count, RDW, platelet count, absolute neutrophil count (ANC), blast count, WHO classification and ferritin. CV was defined as the presence of one or combination of: (a) atherosclerotic hear disease demonstrated by cardiac catheterization; (2) Congestive Heart Failure (CHF); and (3) Peripheral Vascular Disease (PVD). Kaplan-Meier method was used to estimate overall survival (OS). Linear regression model was performed to adjust for multiple variable with potential impact on survival. Results: 44/84 (52.3%) and 40/84 (47.6%) pt were CV positive (pos) and CV negative (neg). Median age for CV disease (pos) and CV (neg) pt was 72 y (range 53-88) and 76 y (range 55-91), respectively, p=0.05. In LR-MDS pt with and without CV disease, median baseline hemoglobin (Hb), RDW, MCV, ferritin and blast count were not statistically different (p>0.05 for all groups). However, baseline ANC and platelet count were 2.1 K/uL vs 1.3 K/uL (p=0.01) and 109 K/uL vs 79 K/uL (p=0.01). Median MCV for all LR-MDS pt was 99 fL. Survival in LR-MDS CV (pos) pt with median MCV > or < 99 fL was 238 vs 634 d, respectively, p= 0.01 (HR 0.6114 95% CI 0.3486 to 1.073). LR-MDS CV (neg) had survival of 831 d vs 382 d (p= 0.3). In LR-MDS CV (pos), Hb at baseline was able to segregate 3 groups with differential survival 290, 659 and 1556 d, respectively, (Hb 10, p=0.1). However, different from CV (pos), hemoglobin segregated 3 distinct survival groups in CV (neg) 301, 430, 1877 d (Hb 10 p= 0.01). RDW as a continuous measure was not predictive for all patients (p=0.085), not for CV (pos) (p=0.908) but predictive for CV (neg) (p=0.012). R-IPSS was significant in all patients (p Conclusion: CV disease is well accepted predictor of poor survival among MDS pt. However, to our knowledge, previous studies examining predictors for outcome in LR-MDS pt diagnosed with CV has not been reported before. MCV is known erythroid parameter frequently abnormal in pt with MDS. Strong correlation between TET2, deregulated IL-1 beta and macrocytosis has been recently uncovered. In our studied population, median MCV specifically discriminated different survival in LR-MDS pt diagnosed with CV disease. Notably, Hb, RDW, platelet count, ferritin and WHO classification reproduced "unique" survival among LR-MDS pt with and without CV disease. Large studies are needed. However, this report emphasizes the need for differential management in LR-MDS CV (pos) vs CV (neg) pt. Disclosures No relevant conflicts of interest to declare.

Background: A number of studies have reported elevated incidence of 25-OH-vitamin D deficiency among patients with multiple myeloma (MM). Several studies have found association between vitamin D levels and factors associated with survival, including ISS stage at diagnosis. However, the impact of vitamin D deficiency on MM prognosis is not entirely clear. Also, in general, both the incidence and the impact of vitamin D deficiency differ substantially by race. Here, we investigate the impact of vitamin D deficiency on prognosis in a large and racially heterogenous patient population with MM in the Veterans Affairs (VA) system. Methods: We used the VA's nationwide Corporate Data Warehouse to identify patients diagnosed with symptomatic MM from 1999 to 2017. Various demographic and laboratory data was collected including age, race, 25-OH-vitamin D levels, and ISS stage at diagnosis as well as survival outcome data. Details of therapies received was also available which indicted similar access to all newer agents approved for myeloma for both African American (AA) and Caucasian patients. Results: We identified 15,717 patients diagnosed with MM (3353 AA and 9070 Caucasian), of whom 6675 had vitamin D measurements within 2 months of diagnosis (1959 AA and 4398 Caucasian). Median serum vitamin D levels were significantly lower among AA patients (21.8 ng/mL) than Caucasians (28.6 ng/mL; p We evaluated the ability of serum vitamin D level to predict overall survival (OS) in patients with MM using a cut-off of 20ng/mL. Patients with vitamin D deficiency ( The analyses were repeated for AA and Caucasian patients separately. Among AA patients, serum vitamin D was not a significant predictor of OS in univariate (P=0.5096) or multivariate analysis (P=0.6923), while it was still a strong predictor among Caucasian patients in both univariate (HR 1.38; P=0.0006) and multivariate analysis (HR 1.45; P=0.0048). Median OS is 3.54 years (95% CI 2.99-5.52; n=255) for AA patients with vitamin D deficiency and 3.95 years (3.25-5.35; n=296) with normal levels. Among Caucasians, median OS is 2.71 years (2.18-3.47; n=273) for deficient and 3.87 years (3.59-4.42; n=885) for normal. Kaplan-Meier plots (Fig. 1B and 1C) illustrate the observed OS curves for the two subgroups. Since levels of vitamin D were lower in AA patients, a lower cut-off of 10 ng/mL was also tested. Even using this lower cutoff, vitamin D deficiency was not a statistically significant predictor of OS in univariate (HR 1.33; P=0.0781) or multivariate analysis (HR 1.09; P=0.7039), though the number of AA patients with vitamin D Conclusions: Vitamin D deficiency is a significant predictor of survival among patients diagnosed with MM, even after accounting for race, age, and ISS stage. However, this relationship is only observed in Caucasian patients and not observed among AA patients. Studies are ongoing to evaluate impact of Vitamin D deficiency of disease presentation including bone disease as well as genetic characteristics. This investigation highlights the need to assess the underlying biological mechanism responsible for the observed impact of vitamin D deficiency across race in MM. Figure 1. Figure 1. Disclosures Yellapragada: Novartis: Employment; Celgene: Research Funding; Takeda: Research Funding. Munshi:OncoPep: Other: Board of director.

Purpose: To assess the safety of administering bortezomib to patients undergoing a radical prostatectomy, to assess pathologic changes induced by bortezomib in prostate cancer specimen, and to verify alterations by the drug in proteasome protein targets. Experimental Design: Bortezomib is a proteasome inhibitor that has shown activity in vitro and in vivo in prostate cancer. We performed a neoadjuvant clinical trial of bortezomib in men with prostate cancer at high risk of recurrence. The primary endpoints were to evaluate safety and biological activity. Results: Bortezomib is generally safe in the preoperative setting. Antitumor activity was manifested by tumor cytopathic effect, drops in serum prostate-specific antigen in some patients, and increases in tumor apoptosis. This was associated with cytoplasmic entrapment of nuclear factor-κB. We found an unexpected increase in proliferation in treated tissues and in vitro. Bortezomib also increased SRC-3 levels and phosphorylated Akt, both in vitro and in treated prostate cancer tissues. Knockdown of SRC-3 blocked the increase in activated Akt in vitro. Combined treatment with bortezomib and the Akt inhibitor perifosine was more effective than either agent alone in vitro. Conclusion: These data suggest that combined therapies targeting the proteasome and the Akt pathway may have increased efficacy.

TMPRSS2-ERG fusion occurs in about half of prostate cancers and results in over-expression of the oncogenic ERG protein in the prostate. The mechanism by which ERG contributes to prostate cancer initiation and progression remains largely unknown. Because ERG is a transcriptional activator, we reasoned that the target genes regulated by ERG could contribute to prostate cancer development.In a search for ERG target genes, we took advantage of published datasets from the MSKCC Prostate Oncogene Project, in which a comprehensive analysis was applied to define transcriptomes in 150 prostate tumors. We retrieved the mRNA expression dataset, split them based on ERG expression, and identified genes whose expression levels are associated with ERG mRNA levels.mRNA expression levels of 21 genes were found to be significantly increased, while for one gene it was decreased in ERG-positive prostate tumors. Among them, the expression of TDRD1 was the most significantly increased in ERG-positive tumors. Among 131 primary prostate tumors which were primarily from European American patients, TDRD1 is over-expressed in 68% of samples, while ERG is overexpressed in 48% of samples, suggesting an additional ERG-independent mechanism of TDRD1 overexpression. In African American prostate tumors, TDRD1 mRNA is expressed in 44%, while ERG is expressed in 24% of samples. In normal tissues, TDRD1 mRNA is exclusively expressed in germ cells and its protein is also known as cancer/testis antigen 41.1 (CT41.1). We generated a mouse monoclonal antibody that recognizes human TDRD1 protein with high specificity and sensitivity. By Western blot analysis and immunohistochemistry (IHC) staining, we demonstrate that TDRD1 protein is expressed in the majority of human prostate tumors, but not in normal prostate tissue. Finally, TDRD1 is not induced in the prostate of ERG overexpression transgenic mice, suggesting that such model does not fully recapitulate the TMPRSS2/ERG fusion-dependent human prostate cancer development.Our results suggest TDRD1 as a novel prostate cancer biomarker. As an ERG target gene, TDRD1 might play an important role in human prostate cancer development, and as a cancer/testis antigen, TDRD1 might have long-term potential to be a therapeutic target for prostate cancer immunotherapy. Prostate 76:1271-1284, 2016. © 2016 Wiley Periodicals, Inc.

Up-regulation of caveolin-1 (cav-1) has been implicated in human prostate cancer progression/metastasis and shown to promote cancer cell survival. It has also been shown that cav-1 is secreted by tumor cells and may regulate the growth, functional activities, and migration of vascular endothelial cells. However, the relationship of cav-1 expression in prostate cancer cells and tumor associated endothelial cells (TAEC) to tumor-associated angiogenesis remains to be investigated. Dual immunofluorescent labeling with antibodies to CD34 and cav-1 was performed on 56 prostate cancer specimens obtained by radical prostatectomy and stratified according to cav-1 positivity in cancer cells. The tumor microvessel densities (MVD) and cav-1 expression in TAEC within these specimens were measured and correlated with cav-1 expression in prostate cancer cells. The MVD values were significantly higher in cav-1-positive (n = 25) than in the cav-1-negative (n = 31) tumors (median of 44 versus 25 vessels/field, P = .0140). Additional studies showed that the cav-1 positivity in microvessels within tumor specimens was significantly less frequent than in the blood vessels of benign prostatic tissues (94.4% versus 98.6%, P = .0012). In contrast, the percentage of cav-1-positive TAEC in cav-1-positive tumors was significantly higher than in cav-1-negative tumors (95.8% versus 92.7%, P = .0024). This increased cav-1 positivity in TAEC was predominantly confined to regions with cav-1-positive tumor cells corresponding to the higher percentage of cav-1-positive microvessels within these regions in cav-1-positive, as opposed to cav-1-negative tumors (P = .0086). These positive correlations provide new evidence for the involvement of prostate cancer cell derived cav-1 in mediating angiogenesis during prostate cancer progression. They also establish a conceptual framework for further investigation of cav-1 proangiogenic activities.

Summary In vitro studies suggest that the proapoptotic function of forkhead protein FKHR is probably inactivated by means of phosphorylation through the protein kinase B pathway. However, the clinical significance of FKHR in prostate cancer remains unclear. Six hundred forty radical prostatectomies were used for building tissue microarrays. Slides were stained with antibodies against FKHR and phosphorylated FKHR (p-FKHR). Correlations with clinicopathologic parameters were analyzed by Spearman rank test. Cox regression test and Kaplan-Meier test were used to determine the probability of disease recurrence, which is defined as a serum prostate-specific antigen (PSA) level greater than 0.4 ng/mL after radical prostatectomy. Nuclear FKHR level was higher in normal prostate than in benign prostatic hyperplasia and prostate cancer ( P = .0000). Nuclear expression of FKHR was correlated with preoperative PSA level ( ρ = 0.108, P = .029), extracapsular extension ( ρ = 0.137, P = .005), and seminal vesicle invasion ( ρ = 0.101, P = .039). FKHR expression was not a significant indicator of biochemical failure by either univariate or multivariate analysis. Nuclear p-FKHR expression correlated with patients' age ( ρ = 0.179, P = .0003), Gleason score ( ρ = 0.130, P = .0083), extracapsular extension ( ρ = 0.227, P = .0000), clinical stage (Union Internationale Contre le Cancer system) ( ρ = 0.166, P = .0007), and lymph node status ( ρ = 0.101, P = .0401). Cytoplasmic p-FKHR correlated with patients' age ( ρ = 0.146, P = .0030) and clinical stage ( ρ = 0.117, P = .0180). Cytoplasmic p-FKHR was a significant indicator of biochemical recurrence ( P = .0164; hazard ratio, 1.114-2.929). Nuclear p-FKHR strongly correlated with phosphorylated protein kinase B ( ρ = 0.368, P = .0000), androgen receptor ( ρ = 0.385, P = .0000), and Skp-2 ( ρ = 0.170, P = .0036). Our data suggest that the proapoptotic role of FKHR is probably regulated by several signaling pathways in prostate cancer.

Prostate cancer is an androgen-dependent disease; metastatic prostate cancer is typically treated by androgen receptor (AR) blockade. Recurrence after androgen ablation and evidence that AR continues to play a role in many prostate cancers has led to an examination of other factors that potentiate AR activity. AR is a ligand-activated transcription factor whose activity is regulated not only by hormone but also by the levels of coactivators recruited by AR to facilitate transcription. We sought to assess the consequences of reducing expression of the transcription intermediary factor 2 (TIF2) coactivator on prostate cancer cell growth and AR action in cell lines to examine TIF2 expression in prostate cancer and to correlate expression with clinical outcome. Depletion of TIF2 reduced expression of AR-induced target genes and slowed proliferation of AR-dependent and AR-independent prostate cancer cells. Remarkably, we found that TIF2 expression is directly repressed by high levels of androgens in multiple AR-expressing cell lines. Expression of a reporter containing 5′-flanking region of the TIF2 was repressed both by androgens and by the antagonist, Casodex. Expression of TIF2 correlates with biochemical (prostate-specific antigen) recurrence (P = 0.0136). In agreement with our in vitro findings, the highest expression of TIF2 was found in patients whose cancer relapsed after androgen ablation therapy, supporting the idea that AR blockade might activate pathways that lead to stimulation of AR-dependent and AR-independent proliferation of prostate epithelium. The elevated expression of TIF2 at low hormone levels likely aids in inducing AR activity under these conditions; treatment with Casodex has the potential to counteract this induction. (Cancer Res 2006; 66(21): 10594-602)

Purpose: Up-regulation of caveolin-1 (cav-1) is associated with virulent prostate cancer, and serum cav-1 levels are elevated in prostate cancer patients but not in benign prostatic hyperplasia. In this study, we evaluated the potential of high preoperative serum cav-1 levels to predict biochemical progression of prostate cancer. The value of the combined preoperative markers, prostate-specific antigen (PSA), biopsy Gleason score, and serum cav-1 for predicting biochemical recurrence was also investigated. Experimental Design: Serum samples taken from 419 prostate cancer patients before radical prostatectomy were selected from our Specialized Programs of Research Excellence prostate cancer serum and tissue bank. Serum samples were obtained 0 to 180 days before surgery and all patients had complete data on age, sex, race, stage at enrollment, and follow-up for biochemical recurrence. Serum cav-1 levels were measured according to our previously reported ELISA protocol. Results: Cav-1 levels were measured in the sera of 419 prostate cancer patients; the mean serum level was 4.52 ng/mL (median 1.01 ng/mL). Patients with high serum cav-1 levels had a 2.7-fold (P = 0.0493) greater risk of developing biochemical recurrence compared with those with low serum cav-1 levels. Importantly, patients with serum PSA ≥ 10 ng/mL and elevated levels of serum cav-1 had 2.44 times higher risk (P = 0.0256) of developing biochemical recurrence compared with patients with low levels of cav-1. In addition, high serum cav-1 levels combined with increasing biopsy Gleason score predicted much shorter recurrence-free survival in the group of patients with PSA ≥ 10 ng/mL (P = 0.0353). Cav-1 was also able to distinguish between high- and low- risk patients with biopsy Gleason score of seven, after adjusting, for patients PSA levels (P = 0.0429). Conclusions: Overall, elevated preoperative levels of serum cav-1 predict decreased time to cancer recurrence. In the subset of patients with serum PSA of ≥10 ng/mL, the combination of serum cav-1 and biopsy Gleason score has the capacity to predict time to biochemical recurrence.

Inappropriate expression of the Aurora kinases can induce aberrant mitosis, centrosome irregularities, and chromosomal instability, which lead to anueploidy and cell transformation. Here, we report that Aurora-A and Aurora-B are highly expressed in primary human and mouse prostate cancers and prostate cancer cell lines. In clinical samples, levels of Aurora-A and Aurora-B were significantly elevated in prostatic intraepithelial neoplasia lesions and prostate tumors when compared with the non-neoplastic samples. Interestingly, expression of Aurora-A in non-neoplastic prostates correlated with seminal vesicle invasion (ρ = 0.275, P = 0.0169) and in prostate tumor with positive surgical margins (ρ = 0.265, P = 0.0161). In addition, nuclear expression of Aurora-B in prostatic intraepithelial neoplasia lesions correlated with clinical staging of the tumor (ρ = −0.4, P = 0.0474) whereas cytoplasmic expression in tumors correlated with seminal vesicle invasion (ρ = 0.282, P = 0.0098). Cell lines and primary tumors derived from the TRAMP model were also found to express high levels of Aurora-A and Aurora-B. When human PC3, LNCaP, and mouse C1A cells were treated with the potent Aurora kinase inhibitor VX680, which attenuates phosphorylation of histone H3, cancer cell survival was reduced. VX680 could further reduce cell viability >2-fold when used in combination with the chemotherapy drug doxorubicin. Our findings support a functional relationship between Aurora kinase expression and prostate cancer and the application of small-molecule inhibitors in therapeutic modalities. (Cancer Res 2006; 66(10): 4996-5002)

The limitations of current forms of prostate cancer therapy have driven researchers to search for new alternatives. Previously we showed cytopathic effect related to HSV-tk in prostate cancer. In this study we present initial results of a neoadjuvant HSV-tk gene therapy trial and address some of the potential mechanistic aspects of its effect in human tissues. We enrolled 23 men with clinically localized prostate cancer but high risk for recurrence in this Phase I-II trial. Intraprostatic viral injections (one to four) were followed by 2 weeks of ganciclovir and prostatectomy 2-4 weeks later. Toxicity was modest. Surgical specimens were embedded fully and whole-mount slides were imaged and analyzed for areas of cytopathic effect. The larger the tumor the greater the cytopathic effect. The effect also seems to be related to areas of high CAR expression. However, the number of injection sites did not influence effect. Local (CD8+ cells and macrophages) and systemic immune response (CD8+ and activated CD8+, IL-12) was increased in patients treated with HSV-tk. Increased apoptosis and decreased microvessel density were also noted in these patients. The results suggest a tumor-specific effect mediated by systemic and local immune response, antiangiogenic effect, and modulation of apoptosis.

BACKGROUND Prostate cancer incidence and mortality rates vary widely among individuals of different ethnic/racial groups. We identified a relationship between a subset of genes and race/ethnicity using gene expression profiling. Estrogen receptor α (ERα) was selected for confirmation due to its plausible biological role in cancer susceptibility. METHODS Quantitative polymerase chain reaction (Q-PCR) was used to verify gene expression results. Protein levels of ERα were determined by quantitative immunohistochemistry in a large-scale tissue microarray study (n = 183). RESULTS ERα was significantly higher in stroma of Hispanic and Asian men than in Caucasian (P

Prostate cancer is initially androgen dependent and there is evidence that androgen receptor continues to play a role in androgen-independent prostate cancer. Androgen receptor activity depends both on the level of androgens and on the level of coactivators that interact with androgen receptor. Our goal was to evaluate the role of the androgen receptor coactivator SRC-1 in prostate cancer progression. Using tissue arrays to measure SRC-1 protein levels, we found that increased SRC-1 expression in clinically localized, androgen-dependent cancer is associated with clinical and pathologic variables of increased tumor aggressiveness. Interestingly, there was variable expression of SRC-1 in normal prostate tissue which correlated with the staining intensity of the corresponding cancer tissue. To test the contribution of SRC-1, we examined its role in androgen-dependent LNCaP and androgen-independent C4-2 prostate cancer cell lines. Using small interfering RNA to reduce expression of androgen receptor, we found that androgen receptor was required both for cell growth and for basal expression of prostate-specific antigen in the androgen-independent C4-2 cell line. Thus, although the cells can grow in an androgen-depleted medium, they remained androgen receptor dependent. Reduction of SRC-1 expression significantly reduced growth and altered androgen receptor target gene regulation in both LNCaP and C4-2 cell lines whereas it had no effect on the growth of the androgen receptor–negative PC-3 and DU145 prostate cancer cell lines. Although the requirement for androgens and androgen receptor in the development of prostate cancer is well established, our study implicates enhanced androgen receptor activity through elevated expression of SRC-1 in the development of more aggressive disease in men with prostate cancer.

Perineural invasion (PNI) is the major mechanism of prostate cancer spread outside the prostate. Apoptotic and proliferation indices were determined in PNI cells using the PNI in vitro model and human PNI in tissue microarrays. RNA was extracted from the PNI model and controls and evaluated by cDNA microarray analysis. Differential expression of candidate genes was confirmed by real-time quantitative PCR, fluorescence, and immunohistochemistry using tissue microarrays. Genistein and BAY 11-7085 were added to the supernatant of cocultures and controls in microchamber cultures. The significance of nuclear factor κB (NFκB) nuclear translocation in human PNI was analyzed using Kaplan-Meier analysis. An increase in proliferation and a decrease in apoptosis were observed in human PNI cells and the PNI model as compared with controls. Three of 15 genes up-regulated in the cDNA microarray were involved in the apoptosis signaling pathway (NFκB), and its downstream targets defender against cell death 1 and PIM-2. The increase was corroborated by real-time quantitative PCR and immunofluorescence. NFκB nuclear translocation was seen in the in vitro model and human tissues, where strong nuclear expression was associated with a decrease in recurrence-free survival. Addition of genistein and BAY 11-7085 resulted in a decrease in NFκB, PIM-2 and defender against cell death 1 as well as a reversal of the inhibition of apoptosis. This is the first description of a biological mechanism and functional significance of PNI. Cancer cells in a perineural location acquire a survival and growth advantage using a NFκB survival pathway. Targeting PNI might help detain local spread of the tumor and influence survival.

Prostate cancer (PCa) is androgen dependent and is regulated by androgen/androgen receptor (AR) signaling pathway. However, the clinical significance of AR is in question. In this regard, we have correlated levels of AR expression with some well-established clinical and pathologic parameters and assessed the prognostic value of AR expression in PCa patients treated with radical prostatectomy.A total of 640 cases treated with radical prostatectomy were used to build tissue microarrays. Normal prostate tissue, benign prostatic hyperplasia, and index tumor were cored in triplicate (0.6 mm). An array (2 mm) of 177 metastatic PCa was built as well. Slides were immunostained with an antibody to AR and Ki-67 and digitized. Correlations between AR expression and clinicopathologic variables were analyzed by the Spearman test. Biochemical recurrence-free survival analysis was performed using Kaplan-Meier analysis, and Cox proportional hazard regression was used to determine the probability of disease recurrence.AR was found in epithelial nuclei of both benign and cancer tissues. AR index was higher in normal prostate tissues than that in PCa and benign prostatic hyperplasia and decreased in metastases than PCa. High level of AR expression was correlated with clinical stage, lymph node status, extracapsular extension, seminal vesicle invasion, and Gleason score. High levels of AR status also correlated with high Ki-67 index (r = 0.211, P = 0.0000). By Kaplan-Meier actuarial model, high expression of AR was predictive of a higher probability of recurrence (P = 0.0046, hazards ratio 2.72 [confidence interval 1.28-4.011]). By multivariate analysis, a high level of AR expression was an independent prognostic indicator of biochemical recurrence-free survival (P = 0.0042; hazards ratio 2.422 [confidence interval 1.32-4.44]).High levels of AR are associated with increased proliferation, markers of aggressive disease and are predictive of decreased biochemical recurrence-free survival independently. This confirms the role of AR in tumor growth and progression in hormonally naive PCa.

BACKGROUND The growth and dissemination of tumors has been associated with angiogenesis, which is regulated by a group of polypeptide factors including vascular endothelial growth factor-C (VEGF-C). VEGF-C binds its receptor, vascular endothelial growth factor receptor-3 (VEGFR-3) to promote growth of tumor-associated lymphatic vessels. METHODS In this study, microarray technology was used to build tissue arrays of normal prostate, benign prostate hyperplasia (BPH) and prostate carcinomas (PCa) using tissues from 640 patients. Slides were sectioned and stained with a polyclonal antibody to VEGFR-3 using a standard immunoperoxidase method and digitized. Immunoreactivity was scored using a 0–3+ semiquantitation scoring system for both intensity and percentage. The sum index was obtained by totaling the scores. RESULTS VEGFR-3 is expressed in normal prostate, BPH, and PCa, but VEGFR-3 expression is up-regulated in PCa. We also found that VEGFR-3 is correlated with pre-operative prostate-specific antigen (Pre-PSA), Gleason score, and lymph node metastasis. The recurrence-free 5-year survival in cases with lower sum index (0–3) was significantly higher than that in cases with higher sum index (4–6) (77.3, 69.6%, respectively, P = 0.037) by Kaplan–Meier actuarial model. CONCLUSIONS Our data suggest that VEGFR-3 expression is associated with tumor progression and may play an important role in facilitating lymphatic spread of PCa; high-level of VEGFR-3 expression in prostate cancer cells increases the risk of biochemical recurrence in prostate cancer patients treated by radical prostatectomy. © 2003 Wiley-Liss, Inc.

BACKGROUND WAP-type four disulfide core (WFDC1)/ps20 is a member of the whey acidic protein family, which includes several serine protease inhibitors. Expression of WFDC1/ps20 was previously demonstrated in the normal human prostate stromal compartment. To further current understanding of the role of WFDC1/ps20 in prostate cancer, altered expression of WFDC1/ps20 protein in prostate cancer was evaluated. METHODS Immunohistochemical staining for WFDC1/ps20 was performed using tissue microarrays. Quantitation was based on the percentage of positive-staining stromal or epithelial cells and staining intensity. Resulting data was analyzed relative to the recurrence-free survival data and additional information for this patient set. RESULTS Decreased stromal expression of WFDC1/ps20 predicted shorter recurrence-free survival time by univariate analysis. Decreased stromal WFDC1/ps20 expression correlated with higher radical prostatectomy Gleason scores, positive surgical margins, extracapsular extension, higher clinical stage, and higher preoperative prostate specific antigen levels. Increased epithelial expression of WFDC1/ps20 also predicted shorter recurrence-free survival times by univariate analysis. Increased epithelial expression of WFDC1/ps20 correlated with higher biopsy and radical prostatectomy Gleason scores, and higher clinical stage. CONCLUSIONS Decreased stromal WFDC1/ps20 expression reflects the evolution of a prostate cancer reactive stroma, while increased epithelial WFDC1/ps20 expression may indicate progression to a more aggressive epithelial phenotype and may indicate an epithelial mesenchymal transition (EMT) process. Further evaluation of WFDC1/ps20 biologic functions will aid in the understanding of this interesting expression profile. © 2004 Wiley-Liss, Inc.

Purpose The intensity-modulated radiation therapy (IMRT) treatment planning system generates tightly constricted isodose lines. It is very important to define the margins that are acceptable in the treatment of prostate cancer to maximize the dose escalation and normal tissue avoidance advantages offered by IMRT. It is necessary to take into account subclinical disease and the potential for extracapsular spread. Organ and patient motion as well as setup errors are variables that must be minimized and defined to avoid underdosing the tumor or overdosing the normal tissues. We have addressed these issues previously. The purpose of the study was twofold: to quantify the radial distance of extracapsular extension in the prostatectomy specimens, and to quantify differences between the pathologic prostate volume (PPV), CT-based gross tumor volume (GTV), and planning target volume (PTV). Methods and materials Two related studies were undertaken. A total of 712 patients underwent prostatectomy between August 1983 and September 1995. Pathologic assessment of the radial distance of extracapsular extension was performed. Shrinkage associated with fixation was accounted for with a linear shrinkage factor. Ten patients had preoperative staging studies including a CT scan of the pelvis. The GTV was outlined and volume determined from these CT scans. The PTV, defined as GTV with a 5-mm margin in all dimensions, was then calculated. The Peacock inverse planning system (NOMOS Corp., Sewickley, PA) was used. The PPV, GTV, and PTV were compared for differences and evaluated for correlation. Results Extracapsular extension (ECE) (i.e., prostatic capsular invasion level 3 [both focal and established]) was found in 299 of 712 patients (42.0%). Measurable disease extending radially outside the prostatic capsule (i.e., ECE level 3 established) was noted in 185 of 712 (26.0%). The median radial extension was 2.0 mm (range 0.50–12.00 mm) outside the prostatic capsule. As a group, 20 of 712 (2.8%) had extracapsular extension of more than 5 mm. In the volumetric comparison and correlation study of the GTV and PTV to the PPV, the average GTV was 2 times larger than the PPV. The average PTV was 4.1 times larger than the PPV. Conclusions This is the largest series in the literature quantitatively assessing prostatic capsular invasion (i.e., the radial extracapsular extension). It is the first report of a comparison of PPV to CT-planned GTV and PTV. Using patient and prostate immobilization, 5 mm of margin to the GTV in this study provided sufficient coverage of the tumor volume based on data gathered from 712 patients. In the absence of prostate immobilization, additional margins of differing amounts depending on the technique employed would have to be placed to account for target, patient, and setup uncertainties. The large mean difference between CT-based estimates of the tumor volume and target volume (GTV+PTV) and PPV added further evidence for adequacy of tumor coverage. Target immobilization, setup error, and coverage of subclinical disease must be addressed carefully before successful implementation of IMRT to maximize its ability to escalate dose and to spare normal tissue simultaneously and safely.

Purpose To evaluate the predictors of xerostomia in the treatment of head-and-neck cancers treated with intensity-modulated radiation therapy (IMRT), using the simultaneous modulated accelerated radiation therapy (SMART) boost technique. Dosimetric parameters of the parotid glands are correlated to subjective salivary gland function. Methods and materials Between January 1996 and June 2000, 30 patients with at least 6 months follow-up were evaluated for subjective xerostomia after being treated definitively for head-and-neck cancer with the SMART boost technique. Threshold limits for the ipsilateral and contralateral parotid glands were 35 Gy and 25 Gy, respectively. Dosimetric parameters to the parotid glands were evaluated. The median follow-up time was 38.5 months (mean 39.9 months). The results of the dosimetric parameters and questionnaire were statistically correlated. Results Xerostomia was assessed with a 10-question subjective salivary gland function questionnaire. The salivary gland function questionnaire (questions 1, 2, 3, 4, 6, and 9) correlated significantly with the dosimetric parameters (mean and maximum doses and volume and percent above tolerance) of the parotid glands. These questions related to overall comfort, eating, and abnormal taste. Questions related to thirst, difficulty with speech or sleep, and the need to carry water daily did not correlate statistically with the dosimetric parameters of the parotid glands. Conclusions Questions regarding overall comfort, eating, and abnormal taste correlated significantly with the dosimetric parameters of the parotid glands. Questions related to thirst, difficulty with speech or sleep, and the need to carry water daily did not correlate statistically with the dosimetric parameters of the parotid glands. Dosimetric sparing of the parotid glands improved subjective xerostomia. IMRT in the treatment of head-and-neck cancer can be exploited to preserve the parotid glands and decrease xerostomia. This is feasible even with an accelerated treatment regimen like the SMART boost. More patients need to be evaluated using IMRT to identify relevant dosimetric parameters.

The androgen receptor (AR) is a key driver of prostate cancer (PC), even in the state of castration-resistant PC (CRPC) and frequently even after treatment with second-line hormonal therapies such as abiraterone and enzalutamide. The persistence of AR activity via both ligand-dependent and ligand-independent mechanisms (including constitutively active AR splice variants) highlights the unmet need for alternative approaches to block AR signaling in CRPC. We investigated the transcription factor GATA-binding protein 2 (GATA2) as a regulator of AR signaling and an actionable therapeutic target in PC. We demonstrate that GATA2 directly promotes expression of both full-length and splice-variant AR, resulting in a strong positive correlation between GATA2 and AR expression in both PC cell lines and patient specimens. Conversely, GATA2 expression is repressed by androgen and AR, suggesting a negative feedback regulatory loop that, upon androgen deprivation, derepresses GATA2 to contribute to AR overexpression in CRPC. Simultaneously, GATA2 is necessary for optimal transcriptional activity of both full-length and splice-variant AR. GATA2 colocalizes with AR and Forkhead box protein A1 on chromatin to enhance recruitment of steroid receptor coactivators and formation of the transcriptional holocomplex. In agreement with these important functions, high GATA2 expression and transcriptional activity predicted worse clinical outcome in PC patients. A GATA2 small molecule inhibitor suppressed the expression and transcriptional function of both full-length and splice-variant AR and exerted potent anticancer activity against PC cell lines. We propose pharmacological inhibition of GATA2 as a first-in-field approach to target AR expression and function and improve outcomes in CRPC.

Objective.—To evaluate blood flow velocity and pulsatility in unilateral migraine without aura during the headache-free period using transcranial Doppler (TCD) sonography. Methods.—Patients with unilateral headache were recruited during the headache-free period. Maximum mean flow velocity (MFV) and pulsatility index (PI) were measured in the middle cerebral (MCA) and basilar arteries. Controls were headache-free individuals without cerebrovascular disease. Results.—Twenty-five patients with right-sided migraine, 25 patients with left-sided migraine, and 19 controls were studied. The MCA PI was higher on the right headache side versus the left headache side (0.97 ± 0.2 versus 0.86 ± 0.1 cm/s, P = .02) and versus controls (0.9 ± 0.2 cm/s, NS). The basilar artery MFV was higher in patients with right-sided headache versus left-sided headache (39.5 ± 5.6 versus 34.7 ± 8.2 cm/s, P = .02) and versus controls (38.2 ± 8 cm/s, NS). No decrease in MFV with age was observed in patients with migraine. Conclusions.—Middle cerebral artery flow pulsatility and basilar artery velocity are higher in patients with right-sided migraine compared with left-sided migraineurs, during the headache-free period. Although these parameters were similar to controls, the differences found during the headache-free period in migraineurs may indicate vascular involvement predisposing to the unilateral headache recurrence.

Interval Newton/Generalized Bisection methods reliably find all numerical solutions within a given domain. Both computational complexity analysis and numerical experiments have shown that solving the corresponding interval linear system generated by interval Newton's methods can be computationally expensive (especially when the nonlinear system is large).

Levels of caveolin-1 (Cav-1) in tumour epithelial cells increase during prostate cancer progression. Conversely, Cav-1 expression in the stroma can decline in advanced and metastatic prostate cancer. In a large cohort of 724 prostate cancers, we observed significantly decreased levels of stromal Cav-1 in concordance with increased Gleason score (p = 0.012). Importantly, reduced expression of Cav-1 in the stroma correlated with reduced relapse-free survival (p = 0.009), suggesting a role for stromal Cav-1 in inhibiting advanced disease. Silencing of Cav-1 by shRNA in WPMY-1 prostate fibroblasts resulted in up-regulation of Akt phosphorylation, and significantly altered expression of genes involved in angiogenesis, invasion, and metastasis, including a > 2.5-fold increase in TGF-β1 and γ-synuclein (SNCG) gene expression. Moreover, silencing of Cav-1 induced migration of prostate cancer cells when stromal cells were used as attractants. Pharmacological inhibition of Akt caused down-regulation of TGF-β1 and SNCG, suggesting that loss of Cav-1 in the stroma can influence Akt-mediated signalling in the tumour microenvironment. Cav-1-depleted stromal cells exhibited increased levels of intracellular cholesterol, a precursor for androgen biosynthesis, steroidogenic enzymes, and testosterone. These findings suggest that loss of Cav-1 in the tumour microenvironment contributes to the metastatic behaviour of tumour cells by a mechanism that involves up-regulation of TGF-β1 and SNCG through Akt activation. They also suggest that intracrine production of androgens, a process relevant to castration resistance, may occur in the stroma.