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Intrinsic and/or acquired resistance to cisplatin is a significant obstacle in the treatment of muscle-invasive bladder cancer. p73, a p53 homolog and determinant of chemosensitivity, is rarely mutated in bladder cancer (BC). However p73 expression and therefore function can be repressed through epigenetic changes. In this study, we sought to identify DNA methylation status of p73, expression of TAp73 isoform, and their role in cisplatin sensitivity in BC. Primary tumor samples from 338 bladder cancer patients showed decreased TAp73 expression in MIBC compared to superficial BC. Low TAp73 protein expression was associated with shorter overall survival. To investigate if the loss of expression was methylation dependent, we utilized Illumina 450K methylation arrays to interrogate over 150 BC patient samples. We found 12 distinct CpGs in the p73 gene locus that were hypermethylated in tumors compared to adjacent normal tissues. Patients with high p73 promoter methylation specifically at CpG site cg07382920 had worse survival. In vitro, treatment with a DNA demethylating agent, decitabine (DAC), decreased TAp73 methylation and upregulated expression in both CR-T24 (cisplatin resistant T24 cells) and wild type T24 cells. Furthermore, treatment with DAC increased cisplatin response in wild type T24 and CR-T24. Our studies indicate that TAp73 expression and P1 promoter methylation, specifically at the cg073892920 site, may have prognostic and diagnostic value in MIBC. In the setting of P1 promoter hypermethylation, DAC could be used as a potentiating agent of cisplatin-based chemotherapy.

Patient engagement in medical decision-making improves patient related outcomes through compliance and patient satisfaction. The Inova Schar Cancer Institute has a weekly molecular tumor board (MTB) to match comprehensive genomic sequencing results with targeted therapies for patients. Primary oncologists extended MTB invitations to their patients. Ultimately, 20 of the 139 patients attended and completed pre- and post MTB surveys. There was a statistically significant change from the pre- to post- survey for the question "I am satisfied with how well informed I am about targeted therapy" with P = 0.016. Patients who attended MTB reported higher levels of satisfaction with their knowledge of targeted therapy after MTB as compared to before. A more holistic method of studying this practice would include sampling a larger patient population and a formal evaluation of the physicians' experience with patients attending.

Signaling through the vitamin D receptor has been shown to be biologically active and important in a number of preclinical studies in prostate and other cancers. Epidemiologic data also indicate that vitamin D signaling may be important in the cause and prognosis of prostate and other cancers. These data indicate that perturbation of vitamin D signaling may be a target for the prevention and treatment of prostate cancer. Large studies of vitamin D supplementation will be required to determine whether these observations can be translated into prevention strategies. This paper reviews the available data in the use of vitamin D compounds in the treatment of prostate cancer. Clinical data are limited which support the use of vitamin D compounds in the management of men with prostate cancer. However, clinical trials guided by existing preclinical data are limited.

The vitamin D receptor (VDR) binds the secosteroid hormone 1,25(OH)2D3 with high affinity and regulates gene programs that control a serum calcium levels, as well as cell proliferation and differentiation. A significant focus has been to exploit the VDR in cancer settings. Although preclinical studies have been strongly encouraging, to date clinical trials have delivered equivocal findings that have paused the clinical translation of these compounds. However, it is entirely possible that mining of genomic data will help to refine precisely what are the key anticancer actions of vitamin D compounds and where these can be used most effectively.

Progress has been slow in systemic management of locally advanced and metastatic bladder cancer over the past 20 years. However, the recent approval of immunotherapy with atezolizumab and nivolumab for second-line salvage therapy may usher in an era of more rapid improvement. Systemic treatment is suboptimal and is an area of substantial unmet medical need. The recent findings from The Cancer Genome Atlas project revealed promising pathways that may be amenable to targeted therapies. Promising results with treatment using vascular endothelial growth factor inhibitors such as ramucirumab, sunitinib or bevacizumab, and human epidermal growth factor receptor 2 targeted therapies, epidermal growth factor receptor inhibitors, and fibroblast growth factor receptor inhibitors, are undergoing clinical trials and are discussed later.

Background Preliminary data suggest that the urinary microbiome may play a role in bladder cancer. Information regarding the most suitable method of collecting urine specimens is needed for the large population studies needed to address this. To compare microbiome metrics resulting from 16S ribosomal RNA gene sequencing between midstream, voided specimens and those obtained at cystoscopy. Methods Adults, with a history of superficial urothelial cell carcinoma (non-muscle invasive bladder cancer) being followed with periodic surveillance cystoscopy had a urine sample collected by a mid-stream, voided technique and then from the bladder at cystoscopy. Urine samples underwent 16S ribosomal RNA gene sequencing on the Illumina MiSeq platform. Results 22 subjects (8 female, 14 male) were included. There was no significant difference in beta diversity (diversity between samples) in all samples between collection methods. However, analysis by sex revealed a difference between voided and cystoscopy samples from the same individual in males (p = 0.006, Adonis test) but not in females (p = 0.317, Adonis test). No differences were seen by collection method in any alpha diversity (diversity within a sample) measurement or differential abundance of taxa. Conclusions Beta diversity of the urine microbiome did differ by collection method for males only. This suggests that the urinary microbiomes of the two collection methods are not equivalent to each other, at least in males, which is the sex that bladder cancer occurs most frequently in. Therefore, the same collection method within a given study should be used.

Background Vitamin D3 (VitD) deficiency is linked to increased incidence and worse survival in bladder cancer (BCa). In addition to cystectomy, patients are treated with cisplatin‐based chemotherapy, however 30%‐50% of patients do not benefit from this treatment. The effects of VitD deficiency on response to chemotherapy remain unknown. Methods To test effects of VitD supplementation on the response to cisplatin we analyzed patient serum VitD levels and correlated that with survival. In vivo, VitD deficient mice were treated with cisplatin, with or without pretreatment with the active VitD metabolite, 1,25 dihydroxyvitamin D3 (1,25D3). Lastly, using BCa cell lines, T24 and RT‐112, the mechanism of action of 1,25D3 and cisplatin combination treatment was determined by apoptosis assays, as well as western blot and RT‐PCR. Results In this study, we determined that low serum 25 hydroxyvitamin D3 (25D3) levels was significantly associated with worse response to cisplatin. Pretreating deficient mice with 1,25D3, reduced tumor volume compared to cisplatin monotherapy. In vitro, 1,25D3 pretreatment increased the apoptotic response to cisplatin. 1,25D3 pretreatment increased expression of TAp73 and its pro‐apoptotic targets, in a VDR dependent manner. VDR and its transcriptional targets were induced after 1,25D3 treatment and further increased after the combination of 1,25D3 and cisplatin in a TAp73 dependent manner. Conclusions Our data suggest that VitD deficiency could be a biomarker for poor response to cisplatin, and pretreating with VitD can increase the apoptotic response to cisplatin through VDR and TAp73 signaling crosstalk.

Background While numerous epidemiologic studies have found an association between higher serum 25-hydroxyvitamin D [25(OH)D] concentrations and lower breast cancer risk, few have assessed this association for concentrations >40 ng/ml. Objective To investigate the relationship between 25(OH)D concentration and breast cancer risk across a broad range of 25(OH)D concentrations among women aged 55 years and older. Methods Analyses used pooled data from two randomized clinical trials (N = 1129, N = 2196) and a prospective cohort (N = 1713) to examine a broad range of 25(OH)D concentrations. The outcome was diagnosis of breast cancer during the observation periods (median: 4.0 years). Three analyses were conducted: 1) Incidence rates were compared according to 25(OH)D concentration from

Bladder cancer is the fourth most commonly diagnosed cancer in men and eighth leading cause of cancer-related death in the US. Epidemiological and experimental studies strongly suggest a role for 1α,25(OH)2D3 in cancer prevention and treatment. The antitumor activities of 1α,25(OH)2D3 are mediated by the induction of cell cycle arrest, apoptosis, differentiation and the inhibition of angiogenesis and metastasis. miRNAs play important regulatory roles in cancer development and progression. However, the role of 1α,25(OH)2D3 in the regulation of miRNA expression and the potential impact in bladder cancer has not been investigated. Therefore, we studied 1α,25(OH)2D3-regulated miRNA expression profiles in human bladder cancer cell line 253J and the highly tumorigenic and metastatic derivative line 253J-BV by miRNA qPCR panels. 253J and 253J-BV cells express endogenous vitamin D receptor (VDR), which can be further induced by 1α,25(OH)2D3. VDR target gene 24-hydroxylase was induced by 1α,25(OH)2D3 in both cell lines, indicating functional 1α,25(OH)2D3 signaling. The miRNA qPCR panel assay results showed that 253J and 253J-BV cells have distinct miRNA expression profiles. Further, 1α,25(OH)2D3 differentially regulated miRNA expression profiles in 253J and 253J-BV cells in a dynamic manner. Pathway analysis of the miRNA target genes revealed distinct patterns of contribution to the molecular functions and biological processes in the two cell lines. In conclusion, 1α,25(OH)2D3 differentially regulates the expression of miRNAs, which may contribute to distinct biological functions, in human bladder 253J and 253J-BV cells. This article is part of a Special Issue entitled '17th Vitamin D Workshop'.

Somatic Structural Variations (SVs) are a complex collection of chromosomal mutations that could directly contribute to carcinogenesis. Next Generation Sequencing (NGS) technology has emerged as the primary means of interrogating the SVs of the cancer genome in recent investigations. Sophisticated computational methods are required to accurately identify the SV events and delineate their breakpoints from the massive amounts of reads generated by a NGS experiment. In this review, we provide an overview of current analytic tools used for SV detection in NGS-based cancer studies. We summarize the features of common SV groups and the primary types of NGS signatures that can be used in SV detection methods. We discuss the principles and key similarities and differences of existing computational programs and comment on unresolved issues related to this research field. The aim of this article is to provide a practical guide of relevant concepts, computational methods, software tools and important factors for analyzing and interpreting NGS data for the detection of SVs in the cancer genome.

// Prashant K. Singh 1 , Leah Preus 2 , Qiang Hu 3 , Li Yan 3 , Mark D. Long 1 , Carl D. Morrison 4 , Mary Nesline 2 , Candace S. Johnson 1 , Shahriar Koochekpour 5 , Manish Kohli 6 , Song Liu 3 , Donald L. Trump 7 , Lara E Sucheston-Campbell 2,* and Moray J. Campbell 1,* 1 Department of Pharmacology & Therapeutics, Roswell Park Cancer Institute, Buffalo, NY 2 Department of Cancer Prevention & Control, Roswell Park Cancer Institute, Buffalo, NY 3 Department of Biostatistics & Bioinformatics, Roswell Park Cancer Institute, Buffalo, NY 4 Department of Pathology, Roswell Park Cancer Institute, Buffalo, NY 5 Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, NY 6 Department of Medical Oncology, Mayo Clinic, Rochester, MN 7 Department of Medicine, Roswell Park Cancer Institute, Buffalo, NY * The authors share position as senior and corresponding authors Correspondence: Moray J. Campbell, email: // Lara E Sucheston-Campbell, email: // Keywords : Prostate cancer, microRNA, biochemical progression, miR-103, miR-125b, miR-222, cancer epigenetics Received : January 13, 2014 Accepted : February 13, 2014 Published : February 13, 2014 Abstract We aimed to identify microRNA (miRNA) expression patterns in the serum of prostate cancer (CaP) patients that predict the risk of early treatment failure following radical prostatectomy (RP). Microarray and Q-RT-PCR analyses identified 43 miRNAs as differentiating disease stages within 14 prostate cell lines and reflectedpublically available patient data. 34 of these miRNA were detectable in the serum of CaP patients. Association with time to biochemical progression was examined in a cohort of CaP patients following RP. A greater than two-fold increase in hazard of biochemical progression associated with altered expression of miR-103, miR-125b and miR-222 ( p

Accurate detection of somatic copy number variations (CNVs) is an essential part of cancer genome analysis, and plays an important role in oncotarget identifications. Next generation sequencing (NGS) holds the promise to revolutionize somatic CNV detection. In this review, we provide an overview of current analytic tools used for CNV detection in NGS-based cancer studies. We summarize the NGS data types used for CNV detection, decipher the principles for data preprocessing, segmentation, and interpretation, and discuss the challenges in somatic CNV detection. This review aims to provide a guide to the analytic tools used in NGS-based cancer CNV studies, and to discuss the important factors that researchers need to consider when analyzing NGS data for somatic CNV detections.

Genetic and epigenetic alterations have been identified as to contribute directly or indirectly to the generation of transitional cell carcinoma of the urinary bladder (TCC-UB). In a comparative fashion much less is known about copy number alterations in TCC-UB, but it appears that amplification of chromosome 6p22 is one of the most frequent changes. Using fluorescence in situ hybridization (FISH) analyses, we evaluated chromosomal 6p22 amplification in a large cohort of bladder cancer patients with complete surgical staging and outcome data. We have also used shRNA knockdown candidate oncogenes in the cell based study. We found that amplification of chromosome 6p22.3 is significantly associated with the muscle-invasive transitional cell carcinoma of the urinary bladder (TCC-UB) (22%) in contrast to superficial TCC-UB (9%) (p=7.2-04). The rate of 6p22.3 amplification in pN>1 patients (32%) is more than twice that in pN0 (16%) patients (p=0.05). Interestingly, we found that 6p22.3 amplification is as twice as high (p=0.0201) in African American (AA) than European American (EA) TCC-UB patients. Moreover, we showed that the expression of some candidate genes (E2F3, CDKAL1 and Sox4) in the 6p22.3 region is highly correlated with the chromosomal amplification. In particular, knockdown of E2F3 inhibits cell proliferation in a 6p22.3-dependent manner, whereas knockdown of CDKAL1 and Sox4 has no effect on cell proliferation. Using gene expression profiling, we further identified some common as well as distinctive subset targets of the E2F3 family members. In summary, our data indicate that E2F3 is a key regulator of cell proliferation in a subset of bladder cancer and the 6p22.3 amplicon is a biomarker of aggressive phenotype in this tumor type.

The active vitamin D hormone 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) plays a major role in regulating calcium homeostasis and bone mineralization. 1,25(OH)2D3 also modulates cellular proliferation and differentiation in a variety of cell types. 24-Hydroxylase, encoded by the CYP24A1 gene, is the key enzyme which converts 1,25(OH)2D3 to less active calcitroic acid. Nearly all cell types express 24-hydroxylase, the highest activity being observed in the kidney. There is increasing evidence linking the incidence and prognosis of certain cancers to low serum 25(OH)D3 levels and high expression of vitamin D 24-hydroxylase, supporting the idea that elevated CYP24A1 expression may stimulate degradation of vitamin D metabolites including 25(OH)D3 and 1,25(OH)2D3. The over expression of CYP24A1 in cancer cells may be a factor affecting 1,25(OH)2D3 bioavailability and anti-proliferative activity pre-clinically and clinically. The combination of 1,25(OH)2D3 with CYP24A1 inhibitors enhances 1,25(OH)2D3 mediated signaling and anti-proliferative effects and may be useful in overcoming effects of aberrant CYP24A1 expression. This article is part of a Special Issue entitled 'Vitamin D Workshop'.

Urothelial tumors represent a spectrum of diseases with a range of prognosis. After a diagnosis is established at any point within the urothelial tract, the patient remains at risk for developing a new lesion at a different or the same location and at a similar or more advanced stage. Continued monitoring for recurrence is an essential part of management, because most recurrences are superficial and can be managed endoscopically. Within each category of disease, more refined methods to determine prognosis and guide management, based on molecular staging, are under development with the goal of optimizing the individual patient's likelihood of cure and chance for organ preservation. For patients with more extensive disease, newer treatments typically involve combined-modality approaches, using recently developed surgical procedures, or three-dimensional treatment planning for more precise delivery of radiation therapy. Although these are not appropriate in all cases, they do offer the promise of an improved quality of life and prolonged survival. Finally, within the category of metastatic disease, a number of new agents have been identified that appear to be superior to those currently considered to be standard therapies. Experts believe, therefore, that the treatment of urothelial tumors will evolve rapidly over the next few years, with improved outcomes for patients at all stages of disease.

Vitamin D has broad range of physiological functions and antitumor effects. 24-Hydroxylase, encoded by the CYP24A1 gene, is the key enzyme for degrading many forms of vitamin D including the most active form, 1,25D3. Inhibition of CYP24A1 enhances 1,25D3 antitumor activity. To isolate regulators of CYP24A1 expression in prostate cancer cells, we established a stable prostate cancer cell line PC3 with CYP24A1 promoter driving luciferase expression to screen a small molecular library for compounds that inhibit CYP24A1 promoter activity. From this screening, we identified, 4,5,6,7-tetrabromobenzimidazole (TBBz), a protein kinase CK2 selective inhibitor as a disruptor of CYP24A1 promoter activity. We show that TBBz inhibits CYP24A1 promoter activity induced by 1,25D3 in prostate cancer cells. In addition, TBBz downregulates endogenous CYP24A1 mRNA level in TBBz-treated PC3 cells. Furthermore, siRNA-mediated CK2 knockdown reduces 1,25D3-induced CYP24A1 mRNA expression in PC3 cells. These results suggest that CK2 contributes to 1,25D3-mediated target gene expression. Finally, inhibition of CK2 by TBBz or CK2 siRNA significantly enhances 1,25D3-mediated antiproliferative effect in vitro and in vivo in a xenograft model. In summary, our findings reveal that protein kinase CK2 is involved in the regulation of CYP24A1 expression by 1,25D3 and CK2 inhibitor enhances 1,25D3-mediated antitumor effect. Cancer Res; 73(7); 2289–97. ©2013 AACR.

Preclinical studies demonstrated antiproliferative synergy of 1,25-D3 (calcitriol) with cisplatin. The goals of this phase I/II study were to determine the recommended phase II dose (RP2D) of 1,25-D3 with cisplatin and docetaxel and its efficacy in metastatic non-small-cell lung cancer.Patients were ≥18 years, PS 0-1 with normal organ function. In the phase I portion, patients received escalating doses of 1,25-D3 intravenously every 21 days prior to docetaxel 75 mg/m(2) and cisplatin 75 mg/m(2) using standard 3 + 3 design, targeting dose-limiting toxicity (DLT) rate33 %. Dose levels of 1,25-D3 were 30, 45, 60, and 80 mcg/m(2). A two-stage design was employed for phase II portion. We correlated CYP24A1 tagSNPs with clinical outcome and 1,25-D3 pharmacokinetics (PK).34 patients were enrolled. At 80 mcg/m(2), 2/4 patients had DLTs of grade 4 neutropenia. Hypercalcemia was not observed. The RP2D of 1,25-D3 was 60 mcg/m(2). Among 20 evaluable phase II patients, there were 2 confirmed, 4 unconfirmed partial responses (PR), and 9 stable disease (SD). Median time to progression was 5.8 months (95 % CI 3.4, 6.5), and median overall survival 8.7 months (95 % CI 7.6, 39.4). CYP24A1 SNP rs3787554 (CT) correlated with disease progression (P = 0.03) and CYP24A1 SNP rs2762939 (CG) trended toward PR/SD (P = 0.08). There was no association between 1,25-D3 PK and CYP24A1 SNPs.The RP2D of 1,25-D3 with docetaxel and cisplatin was 60 mcg/m(2) every 21 days. Pre-specified endpoint of 50 % confirmed RR was not met in the phase II study. Functional SNPs in CYP24A1 may inform future studies individualizing 1,25-D3.

Objectives To evaluate the effect of suboptimal dosing on the outcomes of patients who received neoadjuvant chemotherapy (NAC) and robot-assisted radical cystectomy (RARC). Patients and Methods We retrospectively reviewed 336 consecutive patients with urothelial carcinoma of the bladder who were treated with NAC and RARC at three academic institutions. Outcomes were compared between 3 groups: patients who received optimal NAC; patients who received suboptimal NAC; and those who did not receive NAC. To adjust for potential baseline differences between the three groups, propensity-score-based matching was performed. The suboptimal dose group was defined as those who received fewer than three cycles of cisplatin-based chemotherapy, received decreased dosage, or one's not treated with cisplatin. Primary outcomes analyzed were recurrence-free survival (RFS) and overall survival (OS). Secondary outcomes were perioperative complications and readmissions after RARC. Results Within the cohort after propensity-score matching, 69 patients received optimal dose NAC, 41 received suboptimal NAC and 69 did not receive NAC. Complication rates and readmission rates between the 3 groups did not differ significantly. On multivariable analysis, suboptimal dosing and no NAC were independent predictors of worse RFS (HR: 2.5, 95%CI: 1.2-5.7, p=0.01 and HR 2.4, 95%CI 1.28-5.16, p=0.01) and worse OS (HR 4.5, 95%CI 1.6-15.0, p

Bladder urothelial cancers remain an important urologic cancer with limited treatment options in the locally advanced and metastatic setting. While neoadjuvant chemotherapy for locally advanced muscle-invasive cancers has shown overall survival benefit, clinical uptake in practice have lagged behind. Controversies surrounding adjuvant chemotherapy use are also ongoing. Systemic therapies for metastatic bladder cancer have largely used platinum-based therapies without effective standard second-line therapy options for those who fail, although vinflunine is approved in Europe as a second-line therapy based on a Phase III trial, and most recently, atezolizumab, a checkpoint inhibitor, was approved by the US FDA. Given increasing recognition of mutational signatures expressed in urothelial carcinomas, several promising agents with use of VEGF-targeted therapies, HER2-directed agents and immunotherapies with PD-1/PD-L1 antibodies in various settings are discussed herein.

The antiproliferative and pro-apoptotic effects of 1α,25-dihydroxycholecalciferol (1,25(OH)2D3, 1,25D3, calcitriol) have been demonstrated in various tumor model systems in vitro and in vivo. However, limited antitumor effects of 1,25D3 have been observed in clinical trials. This may be attributed to a variety of factors including overexpression of the primary 1,25D3 degrading enzyme, CYP24A1, in tumors, which would lead to rapid local inactivation of 1,25D3. An alternative strategy for improving the antitumor activity of 1,25D3 involves the combination with a selective CYP24A1 inhibitor. The validity of this approach is supported by numerous preclinical investigations, which demonstrate that CYP24A1 inhibitors suppress 1,25D3 catabolism in tumor cells and increase the effects of 1,25D3 on gene expression and cell growth. Studies are now required to determine whether selective CYP24A1 inhibitors+1,25D3 can be used safely and effectively in patients. CYP24A1 inhibitors plus 1,25D3 can cause dose-limiting toxicity of vitamin D (hypercalcemia) in some patients. Dexamethasone significantly reduces 1,25D3-mediated hypercalcemia and enhances the antitumor activity of 1,25D3, increases VDR-ligand binding, and increases VDR protein expression. Efforts to dissect the mechanisms responsible for CYP24A1 overexpression and combinational effect of 1,25D3/dexamethasone in tumors are underway. Understanding the cross talk between vitamin D receptor (VDR) and glucocorticoid receptor (GR) signaling axes is of crucial importance to the design of new therapies that include 1,25D3 and dexamethasone. Insights gained from these studies are expected to yield novel strategies to improve the efficacy of 1,25D3 treatment.

Vitamin D is a secosteroid hormone that regulates many biological functions in addition to its classical role in maintaining calcium homeostasis and bone metabolism. Vitamin D deficiency appears to predispose individuals to increased risk of developing a number of cancers. Compelling epidemiological and experimental evidence supports a role for vitamin D in cancer prevention and treatment in many types of cancers. Preclinical studies show that 1,25D3, the active metabolite of vitamin D, and its analogs have antitumor effects in vitro and in vivo through multiple mechanisms including the induction of cell cycle arrest, apoptosis, differentiation and the suppression of inflammation, angiogenesis, invasion, and metastasis. 1,25D3 also potentiates the effect of chemotherapeutic agents and other agents in the combination treatment. In this review, the antitumor effects of 1,25D3 and the potential underlying mechanisms will be discussed. The current findings support the application of 1,25D3 in cancer prevention and treatment.

Epigenetic alterations occur in tumor-associated vessels in the tumor microenvironment. Methylation of the CYP24A1 gene promoter differs in endothelial cells isolated from tumors and non-tumor microenvironments in mice. The epigenetic makeup of endothelial cells of human tumor-associated vasculature is unknown due to difficulty of isolating endothelial cells populations from a heterogeneous tissue microenvironment. To ascertain CYP24A1 promoter methylation in tumor-associated endothelium, we utilized laser microdissection guided by CD31 immunohistochemistry to procure endothelial cells from human prostate tumor specimens. Prostate tissues were obtained following robotic radical prostatectomy from men with clinically localized prostate cancer. Adjacent histologically benign prostate tissues were used to compare endothelium from benign versus tumor microenvironments. Sodium bisulfite sequencing of CYP24A1 promoter region showed that the average CYP24A1 promoter methylation in the endothelium was 20% from the tumor microenvironment compared with 8.2% in the benign microenvironment (p < 0.05). A 2-fold to 17-fold increase in CYP24A1 promoter methylation was observed in the prostate tumor endothelium compared with the matched benign prostate endothelium in four patient samples, while CYP24A1 promoter methylation remained unchanged in two patient samples. In addition, there is no correlation of the level of CYP24A1 promoter methylation in prostate tumor-associated endothelium with that of epithelium/stroma. This study demonstrates that the CYP24A1 promoter is methylated in tumor-associated endothelium, indicating that epigenetic alterations in CYP24A1 may play a role in determining the phenotype of tumor-associated vasculature in the prostate tumor microenvironment.

There are substantial preclinical and epidemiologic data that suggest that vitamin D plays a role in the prevention and treatment of cancer. Numerous observational studies have shown that low blood levels of 25(OH) vitamin D (cholecalciferol), estimated by geographical location, diet and activity assessment or measured serum levels are associated with a higher risk of cancer and worse cancer-specific survival as well as numerous morbidities to e.g. cardiovascular disease, stroke, infection, autoimmune disease, and neuromuscular dysfunction among large populations. A considerable number of in vitro and in vivo studies indicate that the most active metabolite of vitamin D – 1,25-dihydroxycholecalciferol or calcitriol – has anti-proliferative, pro-apoptotic, pro-differentiating, and anti-angiogenic properties. Combined treatment of calcitriol and many types of cytotoxic agents has synergistic or at least additive effects. However, clinical trials testing these hypotheses have been less encouraging, though a number of methodological, pharmacological, and pharmaceutical issues confound all trials ever conducted. In order to properly assess the clinical value of vitamin D, its metabolites and analogs in cancer prevention and treatment, more studies are needed.

Vitamin D deficiency in the patients treated for breast cancer is associated with numerous adverse effects (bone loss, arthralgia, and falls). The first aim of this study was to assess vitamin D status, determined by 25-OH vitamin D levels, among women diagnosed with breast cancer according to demographic/clinical variables and bone mineral density (BMD). The second aim of this study was to evaluate the effect of daily low-dose and weekly high-dose vitamin D supplementation on 25-OH vitamin D levels. This retrospective study included 224 women diagnosed with stage 0-III breast cancer who received treatment at the James P. Wilmot Cancer Center at the University of Rochester Medical Center. Total 25-OH vitamin D levels (D(2) + D(3)) were determined at baseline for all participants. Vitamin D deficiency was defined as a 25-OH vitamin D level < 20 ng/ml, insufficiency as 20-31 ng/ml, and sufficiency as ≥32 ng/ml. BMD was assessed during the period between 3 months before and 6 months following the baseline vitamin D assessment. Based on the participants' baseline levels, they received either no supplementation, low-dose supplementation (1,000 IU/day), or high-dose supplementation (≥50,000 IU/week), and 25-OH vitamin D was reassessed in the following 8-16 weeks. Approximately 66.5% had deficient/insufficient vitamin D levels at baseline. Deficiency/insufficiency was more common among non-Caucasians, women with later-stage disease, and those who had previously received radiation therapy (P < 0.05). Breast cancer patients with deficient/insufficient 25-OH vitamin D levels had significantly lower lumbar BMD (P = 0.03). Compared to the no-supplementation group, weekly high-dose supplementation significantly increased 25-OH vitamin D levels, while daily low-dose supplementation did not significantly increase levels. Vitamin D deficiency and insufficiency were common among women with breast cancer and associated with reduced BMD in the spine. Clinicians should carefully consider vitamin D supplementation regimens when treating vitamin D deficiency/insufficiency in breast cancer patients.

BACKGROUND Epidemiologic data suggest that there is an association between vitamin D deficiency and influenza infection. We conducted a prospective influenza vaccination study to determine the influence of vitamin D status on serological response to influenza vaccine in prostate cancer (CaP) patients. METHODS During the 2006–2007 influenza season, CaP patients treated at Roswell Park Cancer Institute were offered vaccination with the trivalent influenza vaccine (Fluzone®, 2006–2007) and sera collected for hemagglutination inhibition (HI) assay titers before and 3 months after vaccination. Response to vaccination was defined as ≥1:40 titer ratio or a fourfold increase in titer at 3 months, against any of the three strains. Serum 25-hydroxyvitamin D (25-D3) levels were measured using DiaSorin 125I radioimmunoassay kits. RESULTS Thirty-five patients with CaP participated in the study. Median baseline 25-D3 level was 44.88 ng/ml (range: 9.16–71.98 ng/ml) Serological response against any of the three strains was noted in 80%. There was a significant effect of baseline 25-D3 level when tested as a continuous variable in relation to serological response (P = 0.0446). All patients in the upper quartile of 25-D3 level responded by mounting a serological response (P = 0.0344). None of the other baseline variables (age, race, chemotherapy status, or white cell count) had an effect on serological response. CONCLUSIONS In this study in CaP patients, a replete vitamin D status was associated with more frequent serological response to influenza vaccine. Prostate 77:368–372, 2011. © 2010 Wiley-Liss, Inc.

Gemcitabine is the standard care chemotherapeutic agent to treat pancreatic cancer. Previously we demonstrated that calcitriol (1, 25-dihydroxycholecalciferol) has significant anti-proliferative effects in vitro and in vivo in multiple tumor models and enhances the activity of a variety of chemotherapeutic agents. We therefore investigated whether calcitriol could potentiate the cytotoxic activity of gemcitabine in the human pancreatic cancer Capan-1 model system. Isobologram analysis revealed that calcitriol and gemcitabine had synergistic antiproliferative effect over a wide range of drug concentrations. Calcitriol did not reduce the cytidine deaminase activity in Capan-1 tumors nor in the livers of Capan-1 tumor bearing mice. Calcitriol and gemcitabine combination promoted apoptosis in Capan-1 cells compared with either agent alone. The combination treatment also increased the activation of caspases-8, -9, -6 and -3 in Capan-1 cells. This result was confirmed by substrate-based caspase activity assay. Akt phosphorylation was reduced by calcitriol and gemcitabine combination treatment compared to single agent treatment. However, ERK1/2 phosphorylation was not modulated by either agent alone or by the combination. Tumor regrowth delay studies showed that calcitriol in combination with gemcitabine resulted in a significant reduction of Capan-1 tumor volume compared to single agent treatment. Our study suggests that calcitriol and gemcitabine in combination promotes caspase-dependent apoptosis, which may contribute to increased anti-tumor activity compared to either agent alone.

Many studies indicate calcitriol has potent anti-tumor activity in different types of cancers. However, high levels of vitamin D can produce hypercalcemia in some patients. Glucocorticoids are used to ameliorate hypercalcemia and to enhance calcitriol anti-tumor activity. Calcitriol in combination with the glucocorticoid dexamethasone (Dex) increased vitamin D receptor (VDR) protein levels and ligand binding in squamous cell carcinoma VII (SCC). In this study we found that both calcitriol and Dex induce VDR- and glucocorticoid receptor (GR)-mediated transcription respectively, indicating both hormone receptors are active in SCC. Pre-treatment with Dex increases VDR-mediated transcription at the human CYP24A1 promoter. Whereas, pre-treatment with other steroid hormones, including dihydrotestosterone and R1881, has no effect on VDR-mediated transcription. Real-time PCR indicates treatment with Dex increases Vdr transcripts in a time-dependent manner, suggesting Dex may directly regulate expression of Vdr. Numerous putative glucocorticoid response elements (GREs) were found in the Vdr gene. Chromatin immuno-precipitation (ChIP) assay demonstrated GR binding at several putative GREs located within the mouse Vdr gene. However, none of the putative GREs studied increase GR-mediated transcription in luciferase reporter assays. In an attempt to identify the response element responsible for Vdr transcript regulation, future studies will continue to analyze newly identified GREs more distal from the Vdr gene promoter.

The immune responses to influenza vaccination in patients with colorectal cancer on surveillance or active chemotherapy have not been previously reported. We conducted a prospective influenza vaccination study to determine the serological immune response rate in patients with colorectal cancer.During the 2006-2007 influenza season, patients with colorectal cancer treated at Roswell Park Cancer Institute were offered vaccination with the trivalent influenza vaccine (Fluzone, 2006-2007). Blood samples for hemagglutination inhibition (HI) assay titers were collected before and 3 months after vaccination. Response to vaccination was determined using an endpoint of ≥ 1:40 HI titer ratio or a fourfold HI increase at 3 months from vaccination. A response in HI to at least one of the 3 strains was considered an immune response.Eighty-five patients with colorectal cancer participated in the study. The immune response in the overall population was 70.6%. No differences in response were noted between the 58 patients on active chemotherapy and the 27 patients on surveillance [Odds Ratio (OR) = 0.78; P = 0.8]. The odds of response did not vary by chemotherapy regimen or by chemotherapy-vaccination timing. HI response in all 3 titers concurrently were low in both the chemotherapy (12.1%) and surveillance groups (11.1%) (OR = 1.10; P = 1).Patients with colorectal cancer mount an immune response to influenza vaccination irrespective of their chemotherapy regimen or timing. However, concurrent responses to all three strains in the individual patient with colorectal cancer are uncommon. The investigation of a booster vaccine in this population is warranted.

Considerable preclinical and epidemiologic data suggest that vitamin D may play a role in the pathogenesis, progression, and therapy for cancer. Numerous epidemiologic studies support the hypothesis that individuals with lower serum vitamin D levels have a higher risk of a number of cancers. Measures of vitamin D level in such studies include both surrogate estimates of vitamin D level (residence in more northern latitudes, history of activity, and sun exposure) as well as measured serum 25(OH) cholecalciferol levels. Perhaps, the most robust of these epidemiologic studies is that of Giovannucci et al, who developed and validated an estimate of serum 25(OH) cholecalciferol level and reported that among >40,000 individuals in the Health Professionals Study, an increase in 25(OH) cholecalciferol level of 62.5 ng/mL was associated with a reduction in the risk of head/neck, esophagus, pancreas cancers, and acute leukemia by >50%. Unfortunately, very limited data are available to indicate whether or not giving vitamin D supplements reduces the risk of cancer. Many preclinical studies indicate that exposing cancer cells, as well as vascular endothelial cells derived from tumors, to high concentrations of active metabolites of vitamin D halts progression through cell cycle, induces apoptosis and will slow or stop the growth of tumors in vivo. There are no data that one type of cancer is more or less susceptible to the effects of vitamin D. Vitamin D also potentiates the antitumor activity of a number of types of cytotoxic anticancer agents in in vivo preclinical models. Vitamin D analogues initiate signaling through a number of important pathways, but the pathway(s) essential to the antitumor activities of vitamin D are unclear. Clinical studies of vitamin D as an antitumor agent have been hampered by the lack of a suitable pharmaceutical preparation for clinical study. All commercially available formulations are inadequate because of the necessity to administer large numbers of caplets and the poor "bioavailability" of calcitriol (the most carefully studied analogue) at these high doses. Preclinical data suggest that high exposures to calcitriol are necessary for the antitumor effects. Clinical data do indicate that high doses of calcitriol (>100 mcg weekly, intravenously, and 0.15 microg /kg weekly, orally) can be given safely. The maximum tolerated dose of calcitriol is unclear. While a 250-patient trial in men with castration-resistant prostate cancer comparing docetaxel (36 mg/sqm weekly) +/- calcitriol 0.15 microg/kg indicated that calcitriol was very safe may have reduced to death rate, an adequately powered (1000 patients) randomized study of weekly docetaxel + calcitriol versus q3 week docetaxel was negative. The limitations of this trial were the unequal chemotherapy arms compared in this study and the failure to use an optimal biologic dose or maximum-tolerated dose of calcitriol. In view of the substantial preclinical and epidemiologic data supporting the potential role of vitamin D in cancer, careful studies to evaluate the impact of vitamin D replacement on the frequency of cancer and the impact of an appropriate dose and schedule of calcitriol or other active vitamin D analogue on the treatment of established cancer are indicated.

OBJECTIVE To assess the frequency of vitamin D deficiency among men with prostate cancer, as considerable epidemiological, in vitro, in vivo and clinical data support an association between vitamin D deficiency and prostate cancer outcome. PATIENTS, SUBJECTS AND METHODS The study included 120 ambulatory men with recurrent prostate cancer and 50 with clinically localized prostate cancer who were evaluated and serum samples assayed for 25-OH vitamin D levels. Then 100 controls (both sexes), matched for age and season of serum sample, were chosen from a prospective serum banking protocol. The relationship between age, body mass index, disease stage, Eastern Cooperative Oncology Group performance status, season and previous therapy on vitamin D status were evaluated using univariate and multivariate analyses. RESULTS The mean 25-OH vitamin D level was 25.9 ng/mL in those with recurrent disease, 27.5 ng/mL in men with clinically localized prostate cancer and 24.5 ng/mL in controls. The frequency of vitamin D deficiency (

Preclinical and clinical evidence support an association between vitamin D deficiency and an increased risk of colorectal cancer. Normal vitamin D status has been linked to favorable health outcomes ranging from decreased risk of osteoporosis to improved cancer mortality. We performed a retrospective study to assess the impact of metastatic disease and chemotherapy treatment on vitamin D status in patients with colorectal cancer residing in Western New York.Patients, 315, with colorectal cancer treated in a single institute were assayed for 25-OH vitamin D. The association of age, gender, primary disease site and stage, body mass index, and chemotherapy with vitamin D status was investigated.Vitamin D deficiency was common among participants with a median 25-OH vitamin D level of 21.3 ng/ml (optimal range 32-100 ng/ml). Primary site of disease and chemotherapy status were associated with very low 25-OH vitamin D levels (or =15 ng/ml) on multivariate analysis. Patients receiving chemotherapy and patients with a rectal primary were 3.7 and 2.6-fold more likely to have severe vitamin D deficiency on multivariate analysis than nonchemotherapy patients and colon cancer primary patients, respectively.Chemotherapy is associated with a significant increase in the risk of severe vitamin D deficiency. Patients with colorectal cancer, especially those receiving chemotherapy, should be considered for aggressive vitamin D replacement strategies.

BACKGROUND Preclinical evidence supports the role of estrogen receptor signaling in prostate cancer. In this trial we investigated the tolerability and efficacy of fulvestrant, a pure estrogen receptor antagonist, in the treatment of castration resistant prostate cancer (CRPC). METHODS Patients with CRPC were enrolled after written informed consent. Fulvestrant was administered by intramuscular injection at a dose of 500 mg on day 0, then 250 mg on day 14, day 28 and monthly thereafter. History, physical examination, serum prostate specific antigen (PSA) levels and toxicity was evaluated monthly. Radiographic studies were repeated every 3 months to assess disease. Treatment was continued until disease progression, unacceptable toxicity, non-compliance or consent withdrawal. RESULTS Twenty patients were enrolled over a period of six months. All patients were Caucasians with median age of 69.5 years [range: 47–85 years]. Sixteen patients (80%) had radiological evidence of metastasis and four patients (20%) had rising PSA as the only evidence of progressive disease. Patients received a median of three treatment cycles of fulvestrant [range: 1–11]. Median time to progression was 4.3 months (95% confidence interval of 3–5.7 months) and median overall survival was 19.4 months (range: 9.9–19.4 months) after a median follow-up of 16 months. No patient showed ≥50% reduction in PSA or radiologic improvement. Few adverse events were noted, none of which were attributed directly to fulvestrant. CONCLUSION Fulvestrant was well tolerated but failed to produce clinical or PSA response in men with CRPC. Prostate 68: 1461–1466, 2008. © 2008 Wiley-Liss, Inc.

Aberrant DNA methylation plays a significant role in nearly all human cancers and may contribute to disease progression to advanced phenotypes. Study of advanced prostate cancer phenotypes in the human disease is hampered by limited availability of tissues. We therefore took advantage of the Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) model to study whether three different phenotypes of TRAMP tumors (PRIM, late-stage primary tumors; AIP, androgen-independent primary tumors; and MET, metastases) displayed specific patterns of CpG island hypermethylation using Restriction Landmark Genomic Scanning. Each tumor phenotype displayed numerous hypermethylation events, with the most homogeneous methylation pattern in AIP and the most heterogeneous pattern in MET. Several loci displayed a phenotype-specific methylation pattern; the most striking pattern being loci methylated at high frequency in PRIM and AIP but rarely in MET. Examination of the mRNA expression of three genes, BC058385, Goosecoid, and Neurexin 2, which exhibited nonpromoter methylation, revealed increased expression associated with downstream methylation. Only methylated samples showed mRNA expression, in which tumor phenotype was a key factor determining the level of expression. The CpG island in the human orthologue of BC058385 was methylated in human AIP but not in primary androgen-stimulated prostate cancer or benign prostate. The clinical data show a proof-of-principle that the TRAMP model can be used to identify targets of aberrant CpG island methylation relevant to human disease. In conclusion, phenotype-specific hypermethylation events were associated with the overexpression of different genes and may provide new markers of prostate tumorigenesis. [Cancer Res 2008;68(11):4173–82]

Epidemiological studies indicate that vitamin D insufficiency could have an aetiological role in various human cancers. Preclinical research indicates that the active metabolite of vitamin D, 1alpha,25(OH)2D3, also known as calcitriol, or vitamin D analogues might have potential as anticancer agents because their administration has antiproliferative effects, can activate apoptotic pathways and inhibit angiogenesis. In addition, 1alpha,25(OH)2D3 potentiates the anticancer effects of many cytotoxic and antiproliferative anticancer agents. Here, we outline the epidemiological, preclinical and clinical studies that support the development of 1alpha,25(OH)2D3 and vitamin D analogues as preventative and therapeutic anticancer agents.

Purpose: In preclinical models, calcitriol and the tyrosine kinase inhibitor gefitinib are synergistic and modulate extracellular signal-regulated kinase (Erk) and Akt pathways. Therefore, we conducted a phase I study of calcitriol and gefitinib to determine the maximum tolerated dose (MTD) of this combination.Experimental Design: Calcitriol was given i.v. over 1 h on weeks 1, 3, and weekly thereafter. Gefitinib was given at a fixed oral daily dose of 250 mg starting at week 2 (day 8). Escalation occurred in cohorts of three patients until the MTD was defined. Pharmacokinetic studies were done for calcitriol and gefitinib. Serial skin biopsies were done to investigate epidermal growth factor receptor (EGFR) pathway pharmacodynamic interactions.Results: Thirty-two patients were treated. Dose-limiting hypercalcemia was noted in two of four patients receiving 96 μg/wk of calcitriol. One of seven patients developed dose-limiting hypercalcemia at the MTD 74 μg/wk calcitriol dose level. The relationship between calcitriol dose and peak serum calcitriol (Cmax) and systemic exposure (AUC) was linear. Mean (±SD) serum calcitriol Cmax at the MTD was 6.68 ± 1.42 ng/mL. Gefitinib treatment inhibited EGFR, Akt, and Erk phosphorylation in the skin. Calcitriol did not have consistent effects on skin EGFR or its downstream elements. The combination of gefitinib and calcitriol did not modulate tumor EGFR pathway in patients with serial tumor biopsies.Conclusions: High doses of weekly i.v. calcitriol can be administered safely in combination with gefitinib. Calcitriol concentrations achieved at the MTD 74 μg calcitriol exceed in vivo concentrations associated with antitumor activity in preclinical models.

Studies of genetic variations in vitamin D-related pathways and breast cancer risk have been conducted mostly in populations of European ancestry, and only sparsely in African Americans (AA), who are known for a high prevalence of vitamin D deficiency. We analyzed 24,445 germline variants in 63 genes from vitamin D-related pathways in the African American Breast Cancer Epidemiology and Risk (AMBER) consortium, including 3,663 breast cancer cases and 4,687 controls. Odds ratios (OR) were derived from logistic regression models for overall breast cancer, by estrogen receptor (ER) status (1,983 ER positive and 1,098 ER negative), and for case-only analyses of ER status. None of the three vitamin D-related pathways were associated with breast cancer risk overall or by ER status. Gene-level analyses identified associations with risk for several genes at a nominal p ≤ 0.05, particularly for ER- breast cancer, including rs4647707 in DDB2. In case-only analyses, vitamin D metabolism and signaling pathways were associated with ER- cancer (pathway-level p = 0.02), driven by a single gene CASR (gene-level p = 0.001). The top SNP in CASR was rs112594756 (p = 7 × 10(-5), gene-wide corrected p = 0.01), followed by a second signal from a nearby SNP rs6799828 (p = 1 × 10(-4), corrected p = 0.03). In summary, several variants in vitamin D pathways were associated with breast cancer risk in AA women. In addition, CASR may be related to tumor ER status, supporting a role of vitamin D or calcium in modifying breast cancer phenotypes.

No predictors of a complete pathologic response (pT0) to neoadjuvant chemotherapy (NAC) in muscle-invasive bladder carcinoma have been established. We performed a retrospective analysis of 50 patients to identify potential predictors. Our results showed that the presence of additional transitional cell variants on pathologic examination (mixed tumors) predicted against pT0, suggesting the avoidance of NAC and its morbidity in these patients with mixed tumors.Randomized trials have supported the use of cisplatin-based neoadjuvant chemotherapy (NAC) in muscle-invasive bladder carcinoma (MIBC) owing to the survival advantage, which has correlated with downstaging of the cancer to pT0. Only 30% to 40% of patients receiving NAC have attained a pT0 response at cystectomy; the remaining have either residual disease or progression. We aimed to identify the factors that could predict a pT0 response to NAC.Of 336 patients who had undergone robotic cystectomy at our institute from May 2007 to March 2014, we identified 50 patients who had undergone NAC for MIBC. We conducted a retrospective study, dividing these 50 patients into 2 groups, those with and without a pT0. Factors, including age, histologic features, hydronephrosis at initial presentation, and chemotherapy type, were examined by both univariate and multivariate logistic regression analysis.Of the 50 patients, 14 (28%) had pT0 at cystectomy, 20 (40%) had progressive disease, and 16 (32%) had residual disease. The median age was 67.5 years, the median glomerular filtration rate at presentation was 87.5 mL/min, the patients had undergone a median of 3 NAC cycles, and the median time from the end of chemotherapy to surgery was 4 weeks. The odds of a pT0 response for pure urothelial carcinoma (UC) were approximately 11 times greater relative to cancers with transitional cell variant histologic features or mixed tumors (odds ratio 0.09, 95% confidence interval 0.021-0.380; P = .0011), including squamous, glandular differentiation, small cell, micropapillary, sarcomatoid, nested component, lymphoepithelioma-like, and plasmacytoid variants.The presence of pure UC favored a pT0 response to NAC compared with those with variant histologic features or mixed tumors. These potential predictors warrant prospective validation to allow the ideal selection of patients for NAC.

Abiraterone, a potent CYP 17 inhibitor, is standard treatment in docetaxel refractory, metastatic castrate resistant prostate cancer (mCRPC). However, in countries where abiraterone has not been approved yet, or for patients who cannot afford it, ketoconazole is used as an alternative CYP 17 inhibitor. Although preclinical data suggests that ketoconazole is a less potent inhibitor of CYP 17, there are limited clinical data comparing both agents. We aimed to compare the clinical effectiveness of abiraterone versus ketoconazole in docetaxel refractory mCRPC.Records from mCRPC patients treated with ketoconazole (international multicenter database, n = 162) were reviewed retrospectively. Twenty-six patients treated post docetaxel were individually matched by clinicopathologic factors to patients treated with abiraterone (national multicenter database, n = 140). We compared the PSA response, biochemical and radiological progression free survival (PFS), and overall survival (OS) between the groups. PFS and OS were determined by Cox regression.The groups were matched by Gleason score, pre-treatment disease extent, ECOG PS, pre-treatment risk category (Keizman, Oncologist 2012). Furthermore, they were balanced regarding other known confounding risk factors. In the groups of abiraterone versus ketoconazole, PSA response was 46% versus 19% (OR 4.3, P = 0.04), median biochemical PFS 7 versus 2 months (HR 1.54, P = 0.02), median radiological PFS 8 versus 2.5 months (HR 1.8, P = 0.043), median OS 19 versus 11 months (HR 0.53, P = 0.79), and treatment interruption d/t severe adverse events 8% (n = 2) versus 31% (n = 8) (OR 0.6, P = 0.023).In docetaxel refractory mCRPC, the outcome of abiraterone treatment may be superior to ketoconazole.

The most active metabolite of vitamin D, calcitriol, is growth inhibitory for various tumor types in vitro and in vivo and inhibits the growth of endothelial cells freshly isolated from tumors [tumor-derived endothelial cells (TDEC)]. We compared the effects of calcitriol on Matrigel-derived endothelial cells (MDEC) and TDEC isolated from Matrigel plugs and squamous cell carcinoma tumors, respectively. TDEC and MDEC expressed vitamin D receptor (VDR) and responded to calcitriol by increasing VDR protein expression. Although no mutations were found in VDR from either cell type, Scatchard plot analysis revealed a higher ligand-binding affinity in TDEC (Kd, 0.26 nmol/L) than MDEC (Kd, 0.65 nmol/L). The VDR signaling axis in both cells was intact as shown using nuclear translocation and 24-hydroxylase promoter-luciferase reporter assays. However, unlike TDEC, MDEC were resistant to calcitriol-induced growth inhibition. Calcitriol (10 nmol/L) resulted in a 12.3% growth inhibition of MDEC compared with 47% in TDEC. In TDEC, calcitriol resulted in induction of G0/G1 arrest (10.75%) and reduction of S-phase cells (6.8%) with induction of p27 and down-regulation of p21 protein expression. Apoptotic effects, determined by Annexin V staining were also observed in calcitriol-treated TDEC (38.6%). Calcitriol caused reduced expression of p-Erk and p-Akt and an increase of poly(ADP-ribose) polymerase and caspase-3 cleavage in TDEC. By contrast, none of these effects on cell cycle or apoptosis were seen in calcitriol-treated MDEC. These results show that TDEC were more sensitive than MDEC to the antiproliferative effects of calcitriol despite apparently normal VDR content and structure of signaling axis in both cell types. (Cancer Res 2006; 66(17): 8565-73)

Purpose: To determine, in peripheral blood monocytes (PBM), whether the enzymatic activities of fructose 1,6-bisphosphatase (FBPase), cytidine deaminase (CDDase) and 24-hydroxylase (CYP24), enzymes regulated by calcitriol are useful pharmacodynamic (PD) measures of calcitriol effects in cancer patients. Methods: Cancer patients enrolled in a phase I clinical trial of calcitriol and carboplatin were studied. Baseline and calcitriol-induced changes in FBPase, CDDase and CYP24 activities were measured in PBM collected before, 6, 24, and 48 h after administration of calcitriol, prior to carboplatin, in doses ranging from 4 to 11 μg daily for 3 consecutive days (QD×3). Normal FBPase, CYP24 and CDDase activities were measured in PBM from untreated healthy volunteers. Results: Baseline activities in PBM from cancer patients and healthy volunteers were (median and range): 1.0 (0.0–43.5) and 4.4 (3.1– 8.2) nmol/min/mg protein for FBPase (P = 0.002); 2.5 (0.9–9.3) and 0.8 (0.4–2.0) fmol/h/106 cells for CYP24 (P = 0.016), and 5.6 (2.5–22.3) and 6.6 (1.1–47.4) nmol/min/mg protein for CDDase (P > 0.05), respectively. All calcitriol doses achieved peak serum calcitriol levels > ×3 the physiological levels, increased cancer patient PBM FBPase activity to normal levels and decreased CDDase activity to undetectable levels within 48 h, with no significant change in CYP24 activity. These enzyme activity changes were not associated with hypercalcemia. Conclusions: Calcitriol treatment-induced increase in FBPase and decrease in CDDase activities in cancer patient PBM are potential early and sensitive non-hypercalcemia PD measures of calcitriol effects.

BACKGROUND. Data suggest that vitamin D plays a role in the treatment and prevention of prostate cancer. The combination of high-dose, intermittent calcitriol (1,25 dihydroxyvitamin D3) plus dexamethasone was studied based on evidence that dexamethasone potentiates the antitumor effects of calcitriol and ameliorates hypercalcemia. METHODS. Oral calcitriol was administered weekly, Monday, Tuesday, and Wednesday (MTW), at a dose of 8 g, for 1 month, at a dose of 10 g every MTW for 1 month, and at a dose of 12 g every MTW thereafter. Dexamethasone at a dose of 4 mg was administered each Sunday, and MTW weekly. Calcium and creatinine were determined weekly and radiographs of the urinary tract were performed every 3 months. All patients were considered evaluable for toxicity. RESULTS. Forty-three men with androgen-independent prostate cancer were entered; 37 received at least 1 month of calcitriol given at a dose of 12 g every day 3 per week. The majority of patients had bone metastases and rising prostatespecific antigen (PSA) levels. All had an Eastern Cooperative Oncology Group performance status of 0 or 1. Eight patients (19%) experienced partial responses by PSA criterion (PSA decline of 50%, persisting for 28 days). Subjective clinical improvement occurred in some patients. Toxicity was minimal: urinary tract stones in 2 patients; and a readily reversible, CTC (v.3.0) Grade 2 creatinine increase in 4 patients. Throughout the study only 4 patients ever had a serum calcium level 11.0 mg/dL and no patient had a calcium level 12.0 mg/dL. CONCLUSIONS. The response rate reported in the current study (19%) was not found to be clearly higher than expected with dexamethasone alone. High-dose intermittent calcitriol plus dexamethasone appears to be safe, feasible, and has antitumor activity. Cancer 2006;106:2136 – 42. © 2006 American Cancer Society.

Background: Intermittent administration allows substantial dose escalation of calcitriol but limited bioavailability of the commercially available formulations at high doses is limiting. In this dose escalation study, we sought to evaluate the tolerability and pharmacokinetics of a single oral dose of DN-101, a high-dose calcitriol formulation. Methods: DN-101 doses were escalated in sequential groups of three to six patients with advanced solid tumors. Dose-limiting toxicity was defined as grade ≥2 hypercalcemia or grade ≥3 persistent treatment-related toxicities. Single-dose administration of 15, 30, 60, 75, 90, 105, 135, and 165 μg was tested. Results: Thirty-eight patients were enrolled in 2002 and 2003. The median age was 70 years (range, 44-91 years). Dose escalation was stopped at the 165 μg level when the number of capsules required at one time reached 11. No dose-limiting toxicities occurred. Transient and self-limited grade 3 toxicities were hyponatremia (2) and proteinuria (1). A dose-proportional increase in peak concentration (Cmax) and area under the concentration curve (AUC) was seen across the full range of DN-101 doses tested. At the 165 μg dose, Cmax was 6.21 ± 1.99 ng/mL, AUC(0-24) was 41.3 ± 9.77 ng h/mL, AUC(0-∞) was 55.4 ± 8.44, and half-life (T1/2) was 16.2 hours. Conclusions: At doses between 15 and 165 μg, DN-101 exhibits linear pharmacokinetics. At 165 μg, DN-101 achieves systemic exposure that is 5- to 8-fold higher than that achieved with commercial formulations of calcitriol, which makes DN-101 comparable to that required for antitumor activity in vivo in a murine squamous cell carcinoma model.

Purpose: Docetaxel, cisplatin, and capecitabine are three active chemotherapeutic agents with different mechanisms of action. This phase I study investigated the feasibility and pharmacokinetics of this combination given on a weekly schedule. Experimental Design: Docetaxel and cisplatin were given i.v. over 30 minutes on days 1 and 8 and capecitabine was given orally bid on days 1 to 14 (every 21 days). Escalation occurred in cohorts of three patients until the maximum tolerated dose was defined. Pharmacokinetics studies of docetaxel and total and ultrafiltrate platinum after cisplatin administration were done on cycle 1 (with capecitabine) and cycle 2 (without capecitabine). Results: Twenty-five patients were enrolled. Two of six patients at dose level 5 had a dose-limiting infection and diarrhea. One of six evaluable patients at dose level 4 (27 mg/m2 docetaxel, 27 mg/m2 cisplatin, 825 mg/m2 capecitabine) had a dose-limiting hypomagnesemia. Pharmacokinetics of docetaxel were similar on cycles 1 and 2. Area under the plasma concentrations versus time curves of total platinum was significantly greater in cycle 2 compared with cycle 1 (P = 0.001). There was no difference in the disposition of docetaxel on cycles 1 and 2. Conclusions: The recommended docetaxel, cisplatin, and capecitabine dose for phase II studies is 27/27/825 mg/m2. The alteration in total and ultrafiltrate platinum disposition on cycle 2 compared with cycle 1 may be inherent to sequential cisplatin administration; however, prior treatment with capecitabine cannot be ruled out as a factor.

We have shown previously that sulforaphane (SFN), a constituent of many edible cruciferous vegetables including broccoli, suppresses growth of prostate cancer cells in culture as well as in vivo by causing apoptosis, but the sequence of events leading to cell death is poorly defined. Using PC-3 and DU145 human prostate cancer cells as a model, we now demonstrate, for the first time, that the initial signal for SFN-induced apoptosis is derived from reactive oxygen species (ROS). Exposure of PC-3 cells to growth-suppressive concentrations of SFN resulted in ROS generation, which was accompanied by disruption of mitochondrial membrane potential, cytosolic release of cytochrome c, and apoptosis. All these effects were significantly blocked on pretreatment with N-acetylcysteine and overexpression of catalase. The SFN-induced ROS generation was significantly attenuated on pretreatment with mitochondrial respiratory chain complex I inhibitors, including diphenyleneiodonium chloride and rotenone. SFN treatment also caused a rapid and significant depletion of GSH levels. Collectively, these observations indicate that SFN-induced ROS generation is probably mediated by a nonmitochondrial mechanism involving GSH depletion as well as a mitochondrial component. Ectopic expression of Bcl-xL, but not Bcl-2, in PC-3 cells offered significant protection against the cell death caused by SFN. In addition, SFN treatment resulted in an increase in the level of Fas, activation of caspase-8, and cleavage of Bid. Furthermore, SV40-immortalized mouse embryonic fibroblasts (MEFs) derived from Bid knock-out mice displayed significant resistance toward SFN-induced apoptosis compared with wild-type MEFs. In conclusion, the results of the present study indicate that SFN-induced apoptosis in prostate cancer cells is initiated by ROS generation and that both intrinsic and extrinsic caspase cascades contribute to the cell death caused by this highly promising cancer chemopreventive agent.

17-(Allylamino)-17-demethoxygeldanamycin (17AAG) is a novel anticancer agent in clinical development. The objectives of this study were to develop a population pharmacokinetic model for 17AAG and its major metabolite, 17AG, and to investigate influences of patient characteristics and biochemical markers on pharmacokinetic parameters estimated for 17AAG and 17AG. In a phase I clinical study, 17AAG was administered by intravenous infusion to 43 patients with refractory, advanced malignancies. Plasma concentrations of 17AAG and 17AG were determined by high-performance liquid chromatography. Plasma concentration vs time data were modeled using NONMEM. Nine covariates (age, sex, performance status, weight, height, body surface area, AST, bilirubin and serum creatinine) were investigated for their influences on individual pharmacokinetic parameters. Plasma concentration vs time data were best described by a two-compartment model for 17AAG and a one-compartment model for 17AG. Volumes of distribution were 24.2 and 89.6 l for 17AAG. Total elimination clearances were 26.7 and 21.3 l/h for 17AAG and 17AG, respectively. Both fixed and random effects pharmacokinetic parameters were well estimated. None of the covariates explained the interindividual variability in 17AAG and 17AG pharmacokinetic parameters or improved the fit of the model based on objective function changes. A population pharmacokinetic model was developed to describe 17AAG and 17AG population pharmacokinetic parameters and interindividual variabilities. There were substantial interindividual variabilities in 17AAG and 17AG pharmacokinetic parameters despite BSA-normalized dosing.

Stat3, a member of the signal transduction and activation of transcription (STAT) family, is a key signal transduction protein that mediates signaling by cytokines, peptide growth factors, and oncoproteins and is constitutively activated in numerous cancers including prostate. Previous studies demonstrated that constitutively activated Stat3 plays an important role in the development and progression of prostate cancer by promoting cell proliferation and protecting against apoptosis. The present study was designed to investigate the potential use of RNA interference to block Stat3 expression and activation and the effect on the growth of human prostate cancer cells. We identified a small interfering RNA (siRNA) specific for Stat3 and demonstrate that blockade of Stat3 activation by the Stat3 siRNA suppresses the growth of human prostate cancer cells and Stat3-mediated gene expression and induces apoptotic cell death. The Stat3 siRNA does not inhibit the proliferation nor induces apoptosis of Stat3-inactive human prostate cancer cells. In addition, the Stat3 siRNA inhibits the levels of androgen-regulated prostate specific antigen (PSA) expression in prostate cancer cells. These results demonstrate that targeting Stat3 signaling using siRNA technique may serve as a novel therapeutic strategy for treatment of prostate cancer expressing constitutively activated Stat3.

Oral administration of 9-nitrocamptothecin (9NC), and the formation of its metabolite 9-aminocamptothecin (9AC), may be associated with high interpatient and intrapatient variability. Therefore, we evaluated the plasma pharmacokinetics and urine recovery of 9NC administered on three different schedules as part of phase I and phase II studies.In phase I schedule A, 9NC was administered orally daily for 5 days per week for 2 weeks repeated every 4 weeks. On phase I schedule B, 9NC was administered daily for 14 days repeated every 4 weeks. In Phase II, 9NC was administered daily for 5 days during 8 weeks (one cycle). Serial blood samples were obtained on day 1 and day 10 or 11 for phase I studies, and day 1 and day 50 for the phase II study. Recovery of 9NC and 9AC in urine was evaluated on day 1 and day 10 or 11 in the phase I study. Area under the 9NC and 9AC plasma concentration vs time curves from 0 to 24 h (AUC0-24 h) were calculated using compartmental analysis.The mean+/-SD 9NC lactone AUC0-24 h values on day 1 at the maximum tolerated dose of schedules A and B (2.43 and 1.70 mg/m2, respectively) and the phase II dose (1.5 mg/m2) were 78.9+/-54.4, 155.7+/-112.8, and 48.3+/-17.5 ng/ml.h, respectively. The mean+/-SD 9AC lactone AUC0-24 h values at these same doses of 9NC were 17.3+/-17.9, 41.3+/-16.6, and 31.3+/-12.8 ng/ml h, respectively. The ratios of 9NC lactone AUC0-24 h on day 10 or 11 to day 1 on phase I A and B were 1.27+/-0.68 and 1.73+/-1.56, respectively, and the ratios 9AC lactone AUC0-24 h on day 10 or 11 to day 1 on phase I A and B were 2.23+/-1.02 and 1.65+/-0.97, respectively. The recovery of 9NC and 9AC in the urine was15%.There was significant interpatient and intrapatient variability in the disposition of 9NC and 9AC. 9NC and 9AC undergo primarily nonrenal elimination.

Purpose: 9-Nitrocamptothecin (9NC) is an oral camptothecin analogue currently administered at 1.5 mg/m2/day × 5 days/week in Phase III studies for pancreatic carcinoma. In an effort to increase the dose administered per day and determine whether the daily dose or number of days of treatment influence toxicity, we performed a Phase I study of 9NC using intermittent schedules of administration.Experimental Design: On schedule A, 9NC was administered orally daily × 5 days for 2 weeks every 4 weeks (one cycle). On schedule B, 9NC was administered orally daily × 14 days every 4 weeks (one cycle). Dose levels were determined by adaptive dose finding. Serial blood samples were obtained on day 1 of each schedule for pharmacokinetic studies of 9NC and its 9-aminocamptothecin (9AC) metabolite, and lactone forms were measured by high-performance liquid chromatography.Results: The recommended Phase II doses for schedules A and B were 2.43 and 1.70 mg/m2/day, respectively, each providing the same dose intensity (i.e., 24 mg/m2/cycle). The primary toxicities on schedules A and B were neutropenia, thrombocytopenia, and diarrhea. On schedule A, two patients with gastric cancer and two patients with pancreatic cancer had stable disease for more than six cycles. On schedule B, one patient with pancreatic cancer had stable disease for more than six cycles, and a patient with pancreatic cancer had a partial response. There was significant interpatient variability in the disposition of 9NC and 9AC. Most of the drug remained in the 9NC form with a ratio of 9NC to 9AC of ∼4 to 1.Conclusions: These studies suggest that 9NC administered on an intermittent schedule is tolerable and may be an active regimen in patients with gastric or pancreatic cancers. Dosing 9NC on a mg/m2 basis does not reduce pharmacokinetic variability.

Garlic-derived organosulfides (OSCs) including diallyl trisulfide (DATS) are highly effective in affording protection against chemically induced cancer in animals. Evidence is also mounting to indicate that some naturally occurring OSCs can suppress proliferation of cancer cells by causing apoptosis, but the sequence of events leading to proapoptotic effect of OSCs is poorly defined. Using PC-3 and DU145 human prostate cancer cells as a model, we now demonstrate that DATS is a significantly more potent apoptosis inducer than diallyl sulfide (DAS) or diallyl disulfide (DADS). DATS-induced apoptosis in PC-3 cells was associated with phosphorylation of Bcl-2, reduced Bcl-2 : Bax interaction, and cleavage of procaspase-9 and -3. Bcl-2 overexpressing PC-3 cells were significantly more resistant to apoptosis induction by DATS compared with vector-transfected control cells. DATS treatment resulted in activation of extracellular-signal regulated kinase 1/2 (ERK1/2) and c-jun N-terminal kinase 1 (JNK1) and/or JNK2, but not p38 mitogen-activated protein kinase. Phosphorylation of Bcl-2 in DATS-treated PC-3 cells was fully blocked in the presence of JNK-specific inhibitor SP600125. Moreover, JNK inhibitor afforded significant protection against DATS-induced apoptosis in both cells. DATS-induced Bcl-2 phosphorylation and apoptosis were partially attenuated by pharmacological inhibition of ERK1/2 using PD98059 or U0126. Overexpression of catalase inhibited DATS-mediated activation of JNK1/2, but not ERK1/2, and apoptosis induction in DU145 cells suggesting involvement of hydrogen peroxide as a second messenger in DATS-induced apoptosis. In conclusion, our data point towards important roles for Bcl-2, JNK and ERK in DATS-induced apoptosis in human prostate cancer cells.

BACKGROUND Despite aggressive local therapy, patients with locally advanced bladder cancer are at significant risk for metastases. We evaluated the ability of neoadjuvant chemotherapy to improve the outcome in patients with locally advanced bladder cancer who were treated with radical cystectomy. METHODS Patients were enrolled if they had muscle-invasive bladder cancer (stage T2 to T4a) and were to be treated with radical cystectomy. They were stratified according to age (less than 65 years vs. 65 years or older) and stage (superficial muscle invasion vs. more extensive disease) and were randomly assigned to radical cystectomy alone or three cycles of methotrexate, vinblastine, doxorubicin, and cisplatin followed by radical cystectomy. RESULTS We enrolled 317 patients over an 11-year period, 10 of whom were found to be ineligible; thus, 154 were assigned to receive surgery alone and 153 to receive combination therapy. According to an intention-to-treat analysis, the median survival among patients assigned to surgery alone was 46 months, as compared with 77 months among patients assigned to combination therapy (P=0.06 by a two-sided stratified log-rank test). In both groups, improved survival was associated with the absence of residual cancer in the cystectomy specimen. Significantly more patients in the combination-therapy group had no residual disease than patients in the cystectomy group (38 percent vs. 15 percent, P