Search

The chemopreventive actions of vitamin D were examined in the N-nitroso-tris-chloroethylurea (NTCU) mouse model, a progressive model of lung squamous cell carcinoma (SCC). SWR/J mice were fed a deficient diet (D) containing no vitamin D3, a sufficient diet (S) containing 2,000 IU/kg vitamin D3, or the same diets in combination with the active metabolite of vitamin D, calcitriol (C; 80 μg/kg, weekly). The percentage (%) of the mucosal surface of large airways occupied by dysplastic lesions was determined in mice after treatment with a total dose of 15 or 25 μmol NTCU (N). After treatment with 15 μmol NTCU, the percentages of the surface of large airways containing high-grade dysplastic (HGD) lesions were vitamin D–deficient + NTCU (DN), 22.7% [P < 0.05 compared with vitamin D–sufficient +NTCU (SN)]; DN + C, 12.3%; SN, 8.7%; and SN + C, 6.6%. The extent of HGD increased with NTCU dose in the DN group. Proliferation, assessed by Ki-67 labeling, increased upon NTCU treatment. The highest Ki-67 labeling index was seen in the DN group. As compared with SN mice, DN mice exhibited a three-fold increase (P < 0.005) in circulating white blood cells (WBC), a 20% (P < 0.05) increase in IL6 levels, and a four-fold (P < 0.005) increase in WBC in bronchial lavages. Thus, vitamin D repletion reduces the progression of premalignant lesions, proliferation, and inflammation, and may thereby suppress development of lung SCC. Further investigations of the chemopreventive effects of vitamin D in lung SCC are warranted. Cancer Prev Res; 8(10); 895–904. ©2015 AACR.

Supplemental Table 1. Percent of Normal Airways, Atypia, Low-grade Dysplasia & High-grade Dysplasia Lesions Present in the Small Airways After NTCU Treatment

Supplemental Figure 1. Controls IHC stains

PDF file - 2492K, Effect of 25D3 in combination with TBBz on CYP24A1 mRNA expression and cell proliferation in PC3 cell.

PDF file - 2596K, CYP24A1 mRNA expression in PC3 cell treated with TBBz and 1,25D3

Supplementary Figure 2 from Phenotype-Specific CpG Island Methylation Events in a Murine Model of Prostate Cancer

Calcitriol, a regulator of calcium homeostasis with antitumor properties, is degraded by the product of the CYP24A1 gene, which is downregulated in human prostate cancer by unknown mechanisms. We found that CYP24A1 expression is inversely correlated with promoter DNA methylation in prostate cancer cell lines. Treatment with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (DAC) activates CYP24A1 expression in prostate cancer cells. In vitro methylation of the CYP24A1 promoter represses its promoter activity. Furthermore, inhibition of histone deacetylases by trichostatin A (TSA) enhances the expression of CYP24A1 in prostate cancer cells. Quantitative chromatin immunoprecipitation-PCR (ChIP-qPCR) reveals that specific histone modifications are associated with the CYP24A1 promoter region. Treatment with TSA increases H3K9ac and H3K4me2 and simultaneously decreases H3K9me2 at the CYP24A1 promoter. ChIP-qPCR assay reveals that treatment with DAC and TSA increases the recruitment of vitamin D receptor to the CYP24A1 promoter. Reverse transcriptase-PCR analysis of paired human prostate samples revealed that CYP24A1 expression is downregulated in prostate malignant lesions compared with adjacent histologically benign lesions. Bisulfite pyrosequencing shows that CYP24A1 gene is hypermethylated in malignant lesions compared with matched benign lesions. Our findings indicate that repression of CYP24A1 gene expression in human prostate cancer cells is mediated in part by promoter DNA methylation and repressive histone modifications. Cancer Res; 70(14); 5953–62. ©2010 AACR.

PDF file - 56K, Prostate tumor and matching normal

Supplementary Figure Legends 1-2 from Phenotype-Specific CpG Island Methylation Events in a Murine Model of Prostate Cancer

Supplementary Table 3 from Phenotype-Specific CpG Island Methylation Events in a Murine Model of Prostate Cancer

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.

Supplementary Figure 1 from Phenotype-Specific CpG Island Methylation Events in a Murine Model of Prostate Cancer

Supplementary Figure 1 from Epigenetic Regulation of Vitamin D 24-Hydroxylase/CYP24A1 in Human Prostate Cancer

Supplementary Table 2 from Phenotype-Specific CpG Island Methylation Events in a Murine Model of Prostate Cancer

Supplementary Table 1 from Phenotype-Specific CpG Island Methylation Events in a Murine Model of Prostate Cancer

Brain metastases are rarely observed in urothelial cancers but confer a uniformly poor prognosis. We sought to evaluate the clinical characteristics and course of brain metastases in four patients with primary urothelial cancer with secondary molecular brain tumor characterization available in 3 patients. Mean age was 68 years. Three of the patients were men. Two patients had both primary upper tract and bladder urothelial cancers, and two had bladder cancer alone. Three patients had initial nephroureterectomy and/or radical cystectomy, with subsequent development of initial distant metastasis at a median of 7.9 months. All four patients received platinum-based systemic therapy following initial metastasis. Median onset of brain metastasis from initial distant metastasis was 11.5 months. Median overall survival (OS) from the diagnosis of brain metastasis was 6 months (range: 3.1 – 12.8 months). Three patients who had comprehensive somatic tumor profiling of the brain metastasis using the Oncomine® panel showed mutations in the APC, TP53, and FGFR3 genes, loss of function in TP53 and BRCA-2, and deletion of MHS2. Our findings suggest brain metastases is a uniformly fatal diagnosis and a need for future exploration of primary tumor and metastatic sequencing in order to better define the role of current therapies for metastatic brain urothelial cancers which remains an area of increased unmet need.

INTRODUCTION AND OBJECTIVE:Prostate cancer (PC) provides an opportunity for secondary chemoprevention in men diagnosed with National Comprehensive Cancer Network very low, low, or favorable interme...

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.

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.

Simple Summary Here we applied a powerful predisposition candidate gene identification strategy to identify rare variants shared by two related bladder cancer cases who were members of pedigrees exhibiting a significant excess of bladder cancers. We sequenced the exomes of pairs of related bladder cancer cases belonging to high-risk bladder cancer pedigrees to identify rare, shared variants shared as candidates for predisposition. A rare, shared variant in ERF was also found to show significant association with bladder cancer risk in an independent population, was present in other prostate cancer-affected members in the pedigree, and showed evidence for altering the function of the associated protein. This evidence supports ERF (ETS2 Repressor Factor) as a bladder and prostate cancer predisposition gene. Abstract Pairs of related bladder cancer cases who belong to pedigrees with an excess of bladder cancer were sequenced to identify rare, shared variants as candidate predisposition variants. Candidate variants were tested for association with bladder cancer risk. A validated variant was assayed for segregation to other related cancer cases, and the predicted protein structure of this variant was analyzed. This study of affected bladder cancer relative pairs from high-risk pedigrees identified 152 bladder cancer predisposition candidate variants. One variant in ERF (ETS Repressing Factor) was significantly associated with bladder cancer risk in an independent population, was observed to segregate with bladder and prostate cancer in relatives, and showed evidence for altering the function of the associated protein. This finding of a rare variant in ERF that is strongly associated with bladder and prostate cancer risk in an extended pedigree both validates ERF as a cancer predisposition gene and shows the continuing value of analyzing affected members of high-risk pedigrees to identify and validate rare cancer predisposition variants.

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.

// Yingyu Ma 1 , Wei Luo 1 , Brittany L. Bunch 1 , Rachel N. Pratt 1 , Donald L. Trump 2 and Candace S. Johnson 1 1 Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, NY, USA 2 Inova Schar Cancer Institute, Falls Church, VA, USA Correspondence to: Yingyu Ma, email: // Keywords : 1,25D 3 , bladder cancer, miR-101-3p, migration, invasion Received : May 17, 2017 Accepted : July 19, 2017 Published : July 27, 2017 Abstract Metastasis is the major cause of bladder cancer death. 1,25D 3 , the active metabolite of vitamin D, has shown anti-metastasis activity in several cancer model systems. However, the role of 1,25D 3 in migration and invasion in bladder cancer is unknown. To investigate whether 1,25D 3 affects migration and invasion, four human bladder cell lines with different reported invasiveness were selected: low-invasive T24 and 253J cells and highly invasive 253J-BV and TCCSUP cells. All of the four bladder cancer cells express endogenous and inducible vitamin D receptor (VDR) as examined by immunoblot analysis. 1,25D 3 had no effect on the proliferation of bladder cancer cells as assessed by MTT assay. In contrast, 1,25D 3 suppressed migration and invasion in the more invasive 253J-BV and TCCSUP cells, but not in the low-invasive 253J and T24 cells using “wound” healing, chemotactic migration and Matrigel-based invasion assays. 1,25D 3 promoted the expression of miR-101-3p and miR-126-3p in 253J-BV cells as examined by qRT-PCR. miR-101-3p inhibitor partially abrogated and pre-miR-101-3p further suppressed the inhibition of 1,25D 3 on migration and invasion in 253J-BV cells. Further, 1,25D 3 enhanced VDR recruitment to the promoter region of miR-101-3p using ChIP-qPCR assay. 1,25D 3 enhanced the promoter activity of miR-101-3p as evaluated by luciferase reporter assay. Taken together, 1,25D 3 suppresses bladder cancer cell migration and invasion in two invasive/migration competent lines but not in two less invasive/motile lines, which is partially through the induction of miR-101-3p expression at the transcriptional level.

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.

Pairs of related bladder cancer cases who belong to pedigrees with an excess of bladder cancer were sequenced to identify rare, shared variants as candidate predisposition variants. Candidate variants were tested for association with bladder cancer risk. A validated variant was assayed for segregation to other related cancer cases, and the predicted protein structure of this variant was analyzed. This study of affected bladder cancer relative pairs from high-risk pedigrees identified 152 bladder cancer predisposition candidate variants. One variant in ERF (ETS Repressing Factor) was significantly associated with bladder cancer risk in an independent population, was observed to segregate with bladder and prostate cancer in relatives, and showed evidence for altering the function of the associated protein. This finding of a rare variant in ERF that is strongly associated with bladder and prostate cancer risk in an extended pedigree both validates ERF as a cancer predisposition gene and shows the continuing value of analyzing affected members of high-risk pedigrees to identify and validate rare cancer predisposition variants.

Background: Pancreatic adenocarcinoma is associated with a very poor prognosis, with a 5 year survival of ∼7.2%. Vitamin D has long been evaluated for benefit as a protective agent and treatment for malignancies. Although cancer incidence and outcomes have been tied to vitamin D levels, there is no clear evidence that supplementation of vitamin D improves outcome in pancreatic cancer to date. Case Presentation: We present a patient who errantly took supratherapeutic doses of vitamin D 50,000 U daily, achieving a serum 25(OH)D level of more than 150 mg/mL, with no appreciable side effects. Conclusion: Her disease was stable for 8 months off of conventional treatment, although it is unclear whether this was related to vitamin D supplementation.

Purpose: To evaluate the efficacy and safety of a novel in situ cancer vaccination method for the treatment of aggressive solid tumors, with an initial focus on metastatic prostate cancer (PCa). Procedure: 27 consecutive patients with metastatic cancers (21 with PCa and 6 with other cancers), were treated by in situ cryosurgical lysis of tumor tissue followed by injection of ipilimumab, pembrolizumab or nivolumab, and sargramostim directly into the zone of lysis. This was followed by 30 daily s.c. injections of sargramostim. Patients received 1 to 3 cycles of the above therapy at intervals of ≥ 1 month. Responses to therapy were assessed by RECIST v.1.1 and for patients with PCa, serum PSA levels. This IRB-approved study, Shulman IRB Protocol #00027107, is a retrospective analysis (with prospective follow-up) of the practice of medicine of two physicians. All patients signed informed consent. Results: 21 patients with progressive metastatic PCa and 6 with other metastatic cancers (2 bladder, 1 pancreatic, 1 colon, 1 melanoma, and 1 unknown) were treated. RECIST responses for 2 patients (both with PCa) could not be evaluated due to a lack of follow-up imaging. Among the remaining 25 patients, CRs were seen in 9 (36%) patients and a PR in 1 (4%), for an ORR of 40%. SD was seen in 8 (32%) patients, and progression was seen in 7 (28%). Among the 19 evaluable PCa patients, there were 9 (47%) CRs and no PRs, for an ORR of 47%. 5 (26%) patients showed SD, and 5 (26%) progressed. 13/21 (62%) of patients had post-therapy PSA reductions of >50%. 12 PCa patients were ADT-naïve (11 evaluable by RECIST) and there were 9 with mCRPC (8 evaluable by RECIST), and positive responses were seen in both groups, with ORRs of 55% and 38%, respectively, and PSA reductions of > 50% in 75% and 44% of patients, respectively. These antitumor responses have been durable in many patients, with CRs to date ranging from 1 to over 4.5 years post-treatment. Encouragingly, this durability of response was observed both in ADT-naïve patients and in those with mCRPC. Therapy was well tolerated, with AEs in 19/27 (70%) of patients. 24 grade 1-2 AEs were seen in 19 (70%) patients, and 8 grade 3-4 AEs were seen in 5 (19%) patients. Notably, AEs included liver enzyme elevations, hyperthyroidism and hypothyroidism, all of which are associated with autoimmune responses to immunotherapy. One death that was possibly treatment-related, 4 disease-related deaths, and 2 unrelated deaths occurred. Conclusions: This report describes a novel therapeutic modality utilizing local cryosurgical tumor cell lysis and intratumorally-delivered immunotherapy to treat metastatic prostate cancer and other aggressive solid tumor cancers. Its combination of striking efficacy and good tolerability supports additional formal clinical studies. Citation Format: Gary Onik, David Bostwick, David J. Vaughan, Donald L. Trump, Zurizaday Vega, Timothy Murphy, James Miessau, Marlene Wright-Barton, Danielle Hobbs, Charles J. Link, Jon H. Condra. Regression of metastatic cancer and abscopal effects following in situ vaccination by cryosurgical tumor cell lysis and intratumoral immunotherapy: A case series [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6540.

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

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.

The chemopreventive actions of vitamin D were examined in the N-nitroso-tris-chloroethylurea (NTCU) mouse model, a progressive model of lung squamous cell carcinoma (SCC). SWR/J mice were fed a deficient diet (D) containing no vitamin D3, a sufficient diet (S) containing 2,000 IU/kg vitamin D3, or the same diets in combination with the active metabolite of vitamin D, calcitriol (C; 80 μg/kg, weekly). The percentage (%) of the mucosal surface of large airways occupied by dysplastic lesions was determined in mice after treatment with a total dose of 15 or 25 μmol NTCU (N). After treatment with 15 μmol NTCU, the percentages of the surface of large airways containing high-grade dysplastic (HGD) lesions were vitamin D–deficient + NTCU (DN), 22.7% [P < 0.05 compared with vitamin D–sufficient +NTCU (SN)]; DN + C, 12.3%; SN, 8.7%; and SN + C, 6.6%. The extent of HGD increased with NTCU dose in the DN group. Proliferation, assessed by Ki-67 labeling, increased upon NTCU treatment. The highest Ki-67 labeling index was seen in the DN group. As compared with SN mice, DN mice exhibited a three-fold increase (P < 0.005) in circulating white blood cells (WBC), a 20% (P < 0.05) increase in IL6 levels, and a four-fold (P < 0.005) increase in WBC in bronchial lavages. Thus, vitamin D repletion reduces the progression of premalignant lesions, proliferation, and inflammation, and may thereby suppress development of lung SCC. Further investigations of the chemopreventive effects of vitamin D in lung SCC are warranted. Cancer Prev Res; 8(10); 895–904. ©2015 AACR.

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.

There are considerable data indicating the importance of vitamin D signaling in genitourinary cancers. These studies suggest that vitamin D signaling is a plausible target for the treatment of established cancers—either as vitamin D agents alone or such agents combined with conventional antineoplastic agents. In addition, preclinical and epidemiologic data support the hypothesis that vitamin D supplementation could be a plausible path to genitourinary cancer prevention. Careful studies of vitamin D supplementation will likely be required to determine whether these biologic features can be translated into prevention strategies; similarly, despite several decades of study, there is only limited information regarding the potential role of vitamin D compounds in the treatment of established genitourinary tumors. Two components of further research in this area will be (1) studies of populations of patients to determine factors that may allow investigators to define the molecular characteristics of tumors that would allow investigators to chose those patients most likely to benefit from vitamin D-based therapies. (2) well-designed clinical trials of biologically appropriate doses of vitamin D compounds. Existing data strongly support the continued development of these approaches.

Metastasis is the major cause of bladder cancer death. 1,25D3, the active metabolite of vitamin D, has shown anti-metastasis activity in several cancer model systems. However, the role of 1,25D3 in migration and invasion in bladder cancer is unknown. To investigate whether 1,25D3 affects migration and invasion, four human bladder cell lines with different reported invasiveness were selected: low-invasive T24 and 253J cells and highly invasive 253J-BV and TCCSUP cells. All of the four bladder cancer cells express endogenous and inducible vitamin D receptor (VDR) as examined by immunoblot analysis. 1,25D3 had no effect on the proliferation of bladder cancer cells as assessed by MTT assay. In contrast, 1,25D3 suppressed migration and invasion in the more invasive 253J-BV and TCCSUP cells, but not in the low-invasive 253J and T24 cells using “wound” healing, chemotactic migration and Matrigel-based invasion assays. 1,25D3 promoted the expression of miR-101-3p and miR-126-3p in 253J-BV cells as examined by qRT-PCR. miR-101-3p inhibitor partially abrogated and pre-miR-101-3p further suppressed the inhibition of 1,25D3 on migration and invasion in 253J-BV cells. Further, 1,25D3 enhanced VDR recruitment to the promoter region of miR-101-3p using ChIP-qPCR assay. 1,25D3 enhanced the promoter activity of miR-101-3p as evaluated by luciferase reporter assay. Taken together, 1,25D3 suppresses bladder cancer cell migration and invasion in two invasive/migration competent lines but not in two less invasive/motile lines, which is partially through the induction of miR-101-3p expression at the transcriptional level.

// 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

e18001 Background: Patient engagement in medical decision-making improves patient related outcomes through compliance and patient satisfaction. The Inova Schar Cancer Institute (ISCI) has a weekly molecular tumor board (MTB) to match comprehensive genomic sequencing results with targeted therapies for patients. The ISCI MTB invites patients to attend and engage in the MTB discussion. We performed a pilot study to investigate the feasibility and satisfaction in patients who attended MTB. Methods: During the time of this study, August 2017 through October 2018, 139 patients were presented and all 20 who were able to attend MTB completed pre-and post- MTB surveys. Patients who did not attend were either not invited by their primary oncologist, unable to attend, or chose not to attend. The survey included six questions related to comprehension, engagement, and satisfaction with the treatment team. Results: There was a statistically significant change for the question “I am satisfied with how well informed I am about targeted therapy” with p = 0.016. All 20 patients answered positively that it was beneficial for them to attend. Many patients expressed concerns about their difficulty understanding the technical aspects of the meeting. Conclusions: Patients who attended MTB reported a higher level of satisfaction after MTB attendance as compared to before MTB. This may reflect a sense of engagement in shared decision making rather than comprehension of genomic information. 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. Supported by philanthropic funds from the Inova Schar Cancer Institute.

402 Background: Bladder cancer is estimated to occur in 81,190 patients in 2018 with estimated death in 17,240. Urothelial cancers make up the majority of cancers but non-urothelial cancers are known to have more aggressive phenotype with worse survival. We have demonstrated previously that non-urothelial histologic subtypes have worse survival outcomes compared to urothelial cancers in a population-based analysis (Aragon-Ching J et al.; J Clin Oncol 36, 2018 (suppl 6S: abstr 425). Methods: The primary objective of this review is to gather data on trends and differences between urothelial versus non-urothelial histologies (squamous, sarcomatoid, small cell or neuroendocrine, adenocarcinoma) and compare the demographics, disease characteristics (incidence of metastases, stage presentation, etc), and survival according to the NCDB. Results: Data from diagnosis in year 2004 – 2013 were extracted from the NCDB. A total of 76,858 and 5560 patients were included for urothelial and non-urothelial, respectively. More men were diagnosed with urothelial carcinoma (75%) and non-urothelial carcinoma (61%) than women. The median age was 71 years and 69 years of age, respectively for urothelial and non-urothelial. Majority were Caucasian at 91% and 86%, respectively though there seemed to be more African-Americans in the non-urothelial (11%) compared to 6% in the urothelial cohorts. More patients were diagnosed with higher stages of III and IV in the non-urothelial group (62%) compared to 36% in the urothelial group, although the sites of metastases in the brain, bone or liver were fairly equivalent for both urothelial and non-urothelial cohorts. Median survival time from diagnosis was better in the urothelial compared to non-urothelial arms [69.91 (69.39 – 70.54) vs 64.53 (61.5 – 67.45 months), p-value < 0.001]. Conclusions: Non-urothelial cancers have distinct demographics different from non-urothelial cancers. Patients often present at a higher stage and survival appears to fare worse than urothelial cancers. Further studies to refine treatment options to optimize treatment outcomes are an area of increased unmet need.

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'.

Squamous cell carcinoma of the penis represents approximately 0.5% of all cancers among men in the United States and other developed countries. Although rare, it is associated with significant disfigurement, and only half of the patients survive beyond 5 years. Proper evaluation of both the primary lesion and lymph nodes is critical, because nodal involvement is the most important factor of survival. The NCCN Clinical Practice Guidelines in Oncology for Penile Cancer provide recommendations on the diagnosis and management of this devastating disease based on evidence and expert consensus.