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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.

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.

Hypoxia-inducible factor (HIF) plays an important role in the tumorigenesis of renal cell carcinoma (RCC). Mutational inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene, which is the most frequent molecular event, has been implicated in HIF overexpression. Recent studies found that PIM kinases increase HIF expression in cancer. The PIM Kinases are a family of highly conserved serine/threonine kinases consisting of three isoforms (PIM1, PIM2, and PIM3). PIM kinases play multiple roles in promoting cell proliferation and survival and preventing apoptosis. The expression and the potential function of PIM kinases in RCC remain unclear. Thus, we examined the expression patterns and the clinical significance of the three individual PIM kinases in RCC. PIM expression was examined by immunohistochemical staining tumor tissue and adjacent non-tumor tissue samples from 218 patients with clear cell RCC (ccRCC), including 60 patients with metastatic ccRCC. The expression of each PIM kinase in primary and metastatic ccRCC tissues comparing with adjacent normal tissues was analyzed with variables including tumor stage, grade and survival. Effect of PIM kinase on cell growth in ACHN and 786-O RCC cell lines was examined by individual PIM knockdown with specific siRNA followed by in vitro proliferation assay. Effect of PIM kinase on RCC cell motility was examined by siRNA followed by in vitro Matrigel-based invasion assay. Expression of PIM1, PIM2 and PIM3 was detected in the majority of ccRCC samples (185, 85%), but localization of isoforms differed. PIM1 expression was higher in the nucleus than in cytoplasm, while the reverse was true for PIM 2 and 3. PIM2 and PIM3 expression was higher in cytoplasm than in the nucleus. Higher expression of PIM1 and PIM2 in tumor tissues was significantly associated with metastasis. Nuclear PIM2 was associated with poorly differentiated ccRCC cells. ccRCC patients with increased nuclear PIM2 expression had significantly poorer survival compared with patients with low nuclear PIM2. siRNA-PIM2, but not siRNA-PIM1 and siRNA-PIM3, significantly reduced proliferation of ACHN and 786-O cells. Further, knockdown of PIM2 by siRNA reduced the ability of cell invasion in 786-O cells. In conclusion, PIM kinases, especially PIM2, are associated with ccRCC progression, indicating that PIM kinases may serve as potential markers for metastatic ccRCC. Targeting PIM kinases may have a therapeutic potential in ccRCC. This work was supported by NIH/NCI grant CA067267, CA085142 (Johnson, CS) and National Cancer Institute (NCI) grant P30 CA016056 involving the use of Roswell Park Cancer Institute's Bioinformatics, Pathology Resource Network, Genomics, and Clinical Data Network Shared Resources. Citation Format: Wei Luo, Yingyu Ma, Eduardo Cortes, Angela Omilian, Wiam Bshara, Gissou Azabdaftari, Brittany Bunch, Candace Johnson, Donald Trump. Tumor expression of PIM kinases in renal cell carcinoma and the association with disease progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4625.

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]

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)

Neoadjuvant cisplatin-based chemotherapy is used to treat muscle invasive bladder cancer (MIBC); however the relative 5-year survival of advanced disease is only 15%. The two major pitfalls of neoadjuvant chemotherapy are delaying cystectomy in patients with poor response to cisplatin, and the lack of available biomarkers to identify these patients. Previous studies in our lab showed that pretreatment of preclinical bladder cancer models with 1,25 dihydroxyvitamin D3 (1,25D3), the active metabolite of vitamin D3, enhances the apoptotic response to cisplatin. Mechanistic understanding can provide insight into potential markers for response to 1,25D3 and cisplatin combination therapy in patients. Greater than 50% of MIBCs harbor p53 mutations; however p53 status has not been linked to clinical response to cisplatin. We previously determined that TAp73, a pro-apoptotic p53 family member protein induced after DNA damage, is required for the response to 1,25D3 and cisplatin. In two bladder cancer cell lines with mutant p53, T24 and RT-112, we identified signaling crosstalk between the vitamin D receptor (VDR) and TAp73. Treatment with 1,25D3 and cisplatin, compared to either alone, results in greatest increase of TAp73 mRNA and protein, 2-fold (T24) and 15-fold (RT-112), as well as TAp73 transcriptional target BAX, 3-fold (T24), and 4-fold (RT-112). TAp73 and BAX induction is lost in cells transfected with VDR siRNA, indicating a requirement for VDR. Cells treated with 1,25D3 and cisplatin, compared to either alone, also show the greatest increase of VDR mRNA and protein, 3-fold (T24) and 4-fold (RT-112). CYP24A1, a VDR target gene, has the greatest induction after the combination treatment as well, with an increase of approx. 60-fold (T24), and 100-fold (RT-112). CYP24A1 induction requires TAp73, determined using TAp73 shRNA. These data demonstrate that VDR signaling is enhanced by cisplatin treatment, and TAp73 signaling is enhanced by 1,25D3. We expanded these findings in vivo by treating mice on a vitamin D3 sufficient diet (1,000 IU) or a vitamin D3 deficient diet (25 IU) with 5 mg/kg of cisplatin (IP, q.wk for 3 weeks). Initially, mice on both diets respond to cisplatin treatment, as evidence by a 30% reduction in tumor volume. After the second cycle of treatment, tumors in the deficient mice stopped responding and returned to their initial tumor volume within 24 days. In the sufficient mice, tumors continued to respond to cisplatin with a maximum reduction of 50%. This was maintained for approximately 40 days after treatment. Further study will determine the molecular response to confirm our in vitro mechanism. Our work suggests that vitamin D3 is important in and can increase the response to cisplatin, it also provides rationale for investigating TAp73 protein levels and serum vitamin D3 levels as potential markers to determine patients’ likelihood of responding to cisplatin. Citation Format: Brittany L. Bunch, Donald Trump, Candace S. Johnson. Vitamin D3 increases the response to cisplatin in bladder cancer through VDR and TAp73 pathway crosstalk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1086. doi:10.1158/1538-7445.AM2017-1086

The anti-proliferative and pro-apoptotic effects of 1á,25 dihydroxycholecalciferol (1,25D3) are well-documented in various tumor model systems in vitro and in vivo. However, limited anti-tumor effects of 1,25D3 have been observed in clinical trials. One potential reason may be the over-expression of CYP24A1, the primary 1,25D3 degrading enzyme, in tumors, and the subsequent rapid local inactivation of 1,25D3. A CYP24A1 inhibitor might be added to improve the anti-tumor activity of 1,25D3. However, most CYP24A1 inhibitors are non-specific, and their use is accompanied by a striking increase in the CYP24A1 expression level. In this study, we screened a small molecule library to identify novel CYP24A1 transcriptional inhibitors using a luciferase reporter assay in a human prostate cancer PC3 cell line stably expressing CYP24A1 promoter-driving luciferase reporter. Screening of DIVERSet™, a diverse library of 55,230 compounds, resulted in the identification of 150 hits, each of which had over 50% inhibition of 1,25D3-induced CYP24A1 promoter activity. After confirming initial observations, four hits (CPI3, CPI8, CPI11 and CPI17) displaying the strongest inhibitory effect were chosen to further examine their effects on endogenous and 1,25D3-regulated CYP24A1 expression in cancer cells. Prostate cancer (PC3) cells, bladder cancer (RT112, RT112/D21) cells, kidney cancer (A498, ACHN and 786-O) cells were treated with CPI3, CPI8, CPI11 and CPI17 alone or followed by 100 nM of 1,25D3. qRT-PCR analysis showed that 1,25D3 induced CYP24A1 mRNA expression in these cancer cells and the induction of CYP24A1 was significantly inhibited by the treatment with CPI8 and CPI17 (P < 0.01). Western blot showed the reduction of 1,25D3-induced CYP24A1 protein expression in CPI8 and CPI17 treated cancer cells. These results indicate that CPI8 and CPI17 inhibit CYP24A1 expression at the transcriptional level. To investigate whether these compounds affect the transcriptional expression of vitamin D receptor (VDR) and other vitamin D target genes, the expression of VDR and p21Waf1 were measured by qRT-PCR in kidney cancer A498, ACHN and 786-O cells treated with CPI8, which displayed the strongest inhibitory effect on CYP24A1 expression. Treatment with CPI8 did not affect VDR expression in kidney cancer cells. In contrast with the reduction of CYP24A1 expression by CPI8, treatment with CPI8 markedly increased p21Waf1 mRNA and protein expression compared to 1,25D3 alone. These results indicate that CPI8 differentially affects the expression of vitamin D target genes. In summary, we identified novel CYP24A1 inhibitors, which differentially regulate 1,25D3 target genes. Inhibiting CYP24A1 expression, while increasing the expression of other anti-proliferative 1,25D3-target genes, may be a useful approach to enhance 1,25D3 antitumor activity. Supported by NIH/NCI grant CA67267. Citation Format: Wei Luo, Yingyu Ma, Mikhail Chernov, Donald L. Trump, Candace S. Johnson. Delineation of novel CYP24A1 transcriptional regulators. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 866.

Introduction: African Americans have higher incidence of, and mortality from, prostate cancer (PCa) compared to other racial/ethnic groups. Frequency of polymorphisms in genes involved in vitamin D metabolism, transport, and activity differ by race/ethnicity. Examining the association between polymorphisms in vitamin D-related genes and PCa aggressiveness may explain differing susceptibility to aggressive PCa among individuals and across different racial/ethnic groups. Methods: TagSNPs (n = 315) in 13 genes (VDR, GC, CYP24A1, CYP27A1, CYP27B1, CYP2R1, CYP3A4, DHCR7, CASR, NADSYN1, RXRA, RXRB, RXRG) were genotyped using Illumina GoldenGate or Sequenom assays in 524 African-American and 657 European-American men with newly-diagnosed PCa from PCaP. DNA extracted from blood samples collected at enrollment was stored at -80C prior to analyses. Research subjects were classified as high aggressive if Gleason sum ≥8, or Gleason score (4+3), or PSA >20 ng/ml, or Gleason score (3+4) AND clinical stage = T3-T4. The comparison group (low aggressive) included research subjects with Gleason sum Results: Among African Americans, 21 SNPs were associated with PCa aggressiveness. The variant allele was associated negatively or positively with high aggressive PCa in eleven and ten SNPs, respectively. For example, two SNPs in the vitamin D binding protein gene known as GC, were inversely associated (rs222054: OR, 0.55, 95%CI, 0.38-0.80; and rs16847028: OR, 0.61, 95%CI, 0.39-0.94). Among European Americans, the variant allele was inversely associated with high aggressive PCa for four SNPs in three genes (CASRrs3863977; CYP24A1rs4809960; RXRArs1007971; and RXRArs3118526), and positively associated with high aggressive PCa for three SNPs in the CYP27B1 gene (rs703842, rs4646536, and rs10877013). There was no association between higher number of ‘low vitamin D’ alleles in the four SNPs that comprised the polygenic score and PCa aggressiveness for either race. Conclusions: Polymorphisms in genes involved in vitamin D etabolism and activity, the vitamin D binding protein and calcium sensing receptor were associated with PCa aggressiveness, and there was no overlap in SNPs between race groups. Our ongoing work will examine interaction between polymorphisms of vitamin D-related genes and vitamin D metabolite levels on PCa aggressiveness. Citation Format: Susan E. Steck, Anna Woloszynska-Read, Samuel Antwi, Hongmei Zhang, Lenore Arab, Elizabeth T.H. Fontham, James Mohler, L. Joseph Su, Feifei Xiao, Gary Smith, Donald Trump, Candace Johnson, Jeannette Bensen. SNPs in vitamin D-related genes are associated with prostate cancer aggressiveness in the North Carolina-Louisiana Prostate Cancer Project (PCaP). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 806.

Cisplatin-based combination chemotherapy is the standard treatment for advanced bladder cancer. While there is a substantial initial response rate and occasional complete response, long term control is uncommon. There is a substantial need to develop new therapeutic approaches to bladder cancer. 1,25 dihydroxyvitamin D3 (1,25D3), the active metabolite of vitamin D, enhances the anti-tumor effects of cisplatin in preclinical bladder cancer models. We sought to evaluate the mechanism of 1,25D3 potentiation of cisplatin cytotoxicity through in vitro studies in bladder cancer cell lines. Our studies suggest that enhanced cytotoxicity is mediated through modulation of p73. In a clonogenic assay, the surviving fraction after treatment with 1,25D3 (0.1 uM) for 24 hrs followed by cisplatin (0.1 ug/ml) for 48 hrs increases from 64% to 97% in T24 cells transfected with shRNA targeting p73. The ratio of the pro-apoptotic TAp73 isoform to the anti-apoptotic ΔNp73 isoform is important in determining cellular response to cisplatin. Our studies demonstrate that pretreatment with 1,25D3 followed by cisplatin significantly increases the ratio of TA/ΔNp73 mRNA transcripts 2-fold and increases BAX, the transcriptional target of TAp73, approximately 3-fold. Protein levels of TAp73 and BAX are also increased 2- and 3-fold, respectively, as determined by western blot. Protein stability of TAp73 was determined following cycloheximide treatment for 1, 2, and 4 hrs. After 4 hrs of cycloheximide treatment, protein levels of TAp73 decrease by approximately 50% in all treatment groups except the 1,25D3 and cisplatin group, in which the level of TAp73 protein was maintained. This suggests an increase in TAp73 protein stability after treatment. Using a TransAm p53 binding assay after p53 immunodepletion, 1,25D3 and cisplatin treatment increases DNA binding of TAp73 approximately 2-fold. Protein levels of TAp73 upstream activators, c-Abl and p38, also increase approximately 2-fold after 1,25D3 treatment, suggesting a mechanism of action for increased stability and activation of TAp73. Lastly, pharmacologic inhibition of p38 activation using SB203580 prevents the synergistic effects of 1,25D3 and cisplatin. Taken together, these data suggest that the mechanism of synergy for 1,25D3 and cisplatin combination therapy is through an increase in TAp73 stability and pro-apoptotic transcriptional activity. These findings suggest that 1,25D3 may have potential to be used in combination with cisplatin to increase the apoptotic response. Further studies are being performed to confirm the effects of treatment in an in vivo model, looking both at short term molecular response and long term therapeutic response to treatment. Supported by NCI grants CA067267 and CA016056. Citation Format: Brittany L. Bunch, Candace S. Johnson, Donald L. Trump. Pretreatment with 1,25 dihydroxyvitamin D3 modulates p73 levels and activity to increase pro-apoptotic effects of cisplatin in bladder cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1190.

The Hereditary Cancer Program at Vanderbilt-Ingram Cancer Center (VICC) was established in mid-2012 and provides cancer genetic services to patients and family members who are at risk for family cancer syndromes. During this time, the testing paradigm has markedly shifted from testing a small number of genes to a larger multi-gene set. HYPOTHESIS AND METHODS: We hypothesized that multi-gene testing would identify a higher rate of pathogenic mutations in breast cancer patients than the standard BRCA1/2 testing paradigm. To test this notion, we examined the records of 641 women with breast cancer seen in our clinic from July 2012 through Dec 2014 and tabulated the test outcome in women tested for BRCA1/2 only and women who had multi-gene panels. Patient characteristics were compared between the two groups. RESULTS: Excluding 17 (3%) women with a known familial mutation and the 127 (20%) women who did not proceed with testing, 497 women had usual Sanger BRCA1/2 (189; 38%) or a multi-gene NGS testing (308; 62%). 40 (13%) women were found to have a pathogenic mutation using the multi-gene panel compared to 13 (6%) women who had a restricted BRCA1/2 sequencing (P=0.035, Fishers Exact Test). The 13 women with Sanger BRCA1/2 were younger at diagnosis (40.6 vs 48 yrs) and more likely to have triple negative (TN) disease (38% vs 18%) compared to the 40 women diagnosed with multi-gene panels. TN disease was not confined to BRCA1 carriers, however, as 3 of 7 TN patients had a mutation in BRCA2, PALB2, and ATM, respectively. Thus, 29% (2/7) of our triple negative patients would not have been identified without multi-gene panels. In addition to BRCA1 (6; 15%) and BRCA2 (5; 12.5%), 6 women had mutations in ATM (15%), 7 in CHEK2 (17.5%), 6 in MUTYH (15%), 4 in PALB2 (10%), 2 in TP53 (5%), and 1 each in FANCC, PMS2, RAD51D and XRCC2 (2.5% each). 105 patients (35%) who did not have a deleterious mutation on a multi-gene panel were found to have one or more variants of uncertain significance (VUS) compared to 4 patients who underwent BRCA1/2 testing alone (4/189; 2%). There was a significant difference between providers when ordering hereditary cancer testing, with MD or NP ordering panel testing at a greater rate compared to Genetic Counselors (72% vs 53%; P< 0.0001). CONCLUSION: We have examined the outcomes of genetic tests for 497 women with breast cancer during a time of great change in the approach to testing. Our study supports the paradigm that multi-gene panels will identify additional pathogenic mutations in genes other than BRCA1/2, which could increase clinical efficiency and improve patient outcomes. However, our study also found a high VUS rate in this group of patients, which will require additional clinical time to track for potential changes in pathogenicity. Future studies will focus on potential differences in management for patients found to have alterations on multi-gene tests. Citation Format: Wiesner GL, Lewis S, Holt J, Morgan RH, Riddle DA, Trump SA, McReynolds K. The changing paradigm of hereditary cancer testing: Comparison of tests in 497 women with breast cancer evaluated at an NCI designated cancer center. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-06-01.

Calcitriol (1,25-dihydroxycholecalciferol), the major active form of vitamin D, is antiproliferative in tumor cells and tumor-derived endothelial cells (TDEC). These actions of calcitriol are mediated at least in part by vitamin D receptor (VDR), which is expressed in many tissues including endothelial cells. To investigate the role of VDR in calcitriol effects on tumor vasculature, we established TRAMP-2 tumors subcutaneously into either VDR wild-type (WT) or knockout (KO) mice. Within 30 days post-inoculation, tumors in KO mice were larger than those in WT (P < 0.001). TDEC from WT expressed VDR and were able to transactivate a reporter gene whereas TDEC from KO mice were not. Treatment with calcitriol resulted in growth inhibition in TDEC expressing VDR. However, TDEC from KO mice were relatively resistant, suggesting that calcitriol-mediated growth inhibition on TDEC is VDR-dependent. Further analysis of the TRAMP-C2 tumor sections revealed that the vessels in KO mice were enlarged and had less pericyte coverage compared with WT (P < 0.001). Contrast-enhanced magnetic resonance imaging showed an increase in vascular volume of TRAMP tumors grown in VDR KO mice compared with WT mice (P < 0.001) and FITC-dextran permeability assay suggested a higher extent of vascular leakage in tumors from KO mice. Using ELISA and Western blot analysis, there was an increase of hypoxia-inducible factor-1α, vascular endothelial growth factor, angiopoietin 1, and platelet-derived growth factor-BB levels observed in tumors from KO mice. These results indicate that calcitriol-mediated antiproliferative effects on TDEC are VDR-dependent and loss of VDR can lead to abnormal tumor angiogenesis. [Cancer Res 2009;69(3):967–75]

Bladder cancer is the fourth most common cancer diagnosed and eighth leading cause of cancer death in men. Metastasis is the primary reason for mortality. The 5-year survival rates for localized and metastatic bladder cancers are 94% and 6%, respectively. Therefore, more effective strategies to prevent and inhibit bladder cancer invasion and metastasis are needed to prolong survival. Epidemiological and experimental studies support a role for 1,25(OH) 2D3 (1,25D3) in cancer prevention and treatment. More recently, 1,25D3 has also been reported to inhibit tumor cell invasion and metastasis in several model systems. To study the anti-tumor effect of 1,25D3 in bladder cancer, we selected four human bladder cell lines including T24, 253J, 253J-BV and TCC-SUP cells. 253J-BV is a metastatic line generated from 253J cells through a series of orthotopic inoculation. Vitamin D receptor was expressed and induced by 1,25D3 in all four cell lines. However, treatment with 1,25D3 did not significantly affect the proliferation of the bladder cancer cell lines. To investigate the impact of 1,25D3 on bladder cancer cell migration and invasion, the cells were treated with control EtOH or 1,25D3 in the wound healing assay or chamber-based migration or invasion assays. 1,25D3 suppressed migration and invasion of 253J-BV and TCC-SUP cells but neither 253J nor T24 cells. MiRNAs play critical regulatory roles in cancer cell migration and invasion. We selected 253J and 253J-BV cells for further investigation of the role of miRNAs in 1,25D3 regulation of migration and invasion. Our previous PCR panel studies showed that 1,25D3 differentially regulated miRNA expression profiles in 253J and 253J-BV cells. From the markedly regulated miRNAs, we chose miR-101-3p and miR-126-3p to examine their potential contribution to the inhibition of migration and invasion by 1,25D3. 1,25D3 induced the expression of miR-101-3p and reduced miR-126-3p expression in 253J-BV cells but not 253J cells. To investigate the role of the two miRNAs, we employed pre-miR and miRNA inhibitors. Transfection with miR-101-3p inhibitor vector suppressed migration and invasion in 253J-BV cells. In addition, miR-101-3p inhibitor rescued 1,25D3-induced inhibition of migration and invasion in 253J-BV cells. On the other hand, pre-miR-101-3p transfection followed by 1,25D3 treatment resulted in further reduction of migration and invasion in 253J-BV cells. However, transfection with miR-126-3p inhibitor or pre-miR-126-3p did not alter 1,25D3-inhibition of migration and invasion in 253J-BV cells. These results indicate that 1,25D3 suppresses migration and invasion through the induction of miR-101-3p in human bladder cancer 253J-BV cells. Citation Format: Yingyu Ma, Wei Luo, Rachel Pratt, Donald L. Trump, Candace S. Johnson. 1,25D3 inhibits migration and invasion through miR-101-3p in human bladder cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5095. doi:10.1158/1538-7445.AM2015-5095

Cisplatin-based combination chemotherapy is the standard approach to therapy of advanced bladder cancer. While there is a substantial initial response rate and occasional complete response, long term control is not common. There is a need to develop new therapeutic approaches to bladder cancer. 1,25 dihydroxyvitamin D3 (1,25D3), the active metabolite of vitamin D, enhances the anti-tumor effects of cisplatin in preclinical bladder cancer models. We evaluated the mechanism of 1,25D3 potentiation of cisplatin cytotoxicity through in vitro studies in bladder cancer cell lines. Our studies suggest that enhanced cytotoxicity is mediated through modulation of p73. The ratio of the pro-apoptotic TAp73 isoform to the anti-apoptotic ΔNp73 isoform is important in determining cellular response to cisplatin. Our studies demonstrate that pretreatment with 1,25D3 (0.5 uM) for 24 hrs followed by cisplatin (0.75 ug/ml) for 48 hrs increases the ratio of TA/ΔNp73 mRNA transcripts 2-fold in the T24 bladder cancer cell line, and increases TAp73's transcriptional target, BAX approximately 3-fold compared to cisplatin alone. Protein levels of p73 and BAX, are also increased with 1,25D3 pretreatment followed by cisplatin in T24 cells as determined by western blot. Using a TransAm p53 binding assay after p53 immunodepletion, cisplatin treatment was found to decrease TAp73 functional ability to bind DNA by approximately half, although this has not yet been determined to be statistically significant. 1,25D3 pretreatment followed by cisplatin prevents the decrease in TAp73 DNA binding found after cisplatin alone. These data suggest an increase in TAp73 pro-apoptotic functional abilities resulting from 1,25D3 pretreatment followed by cisplatin in T24 cells. These findings suggest that 1,25D3 may have potential to be used in combination with cisplatin to increase the apoptotic response. Further studies are being performed to determine the requirement of TAp73 in 1,25D3 potentiation of cisplatin cytotoxicity by using T24 cells transfected with TAp73 shRNA in assays previously used to determine synergism, ie. MTT, clonogenic, and apoptosis assays. Supported by NCI grants CA067267 and CA016056. Citation Format: Brittany L. Bunch, Anna Woloszynska-Read, Donald L. Trump, Candace S. Johnson. 1,25 dihydroxyvitamin D3 and cisplatin combination modulate p73 in bladder cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-039. doi:10.1158/1538-7445.AM2015-LB-039

Dysregulation of vitamin D signaling and metabolism enzymes is a frequent change in many cancers and may confound approaches to vitamin -D based therapies. The mechanisms by which aberrant vitamin D signaling and metabolism lead to resistance to vitamin D action are poorly understood. The kidney plays a critical role in vitamin D synthesis and metabolism; in renal cell carcinoma (RCC) cells vitamin D metabolism is dysregulated. Recently, we discovered that an oncogenic protein kinase PIM-1 is involved in vitamin D-induced expression of 24-hydroxylase, a key catalytic enzyme of vitamin D encoded by CYP24A1 gene, in bladder and prostate cancer. To study the impact of PIM1 on vitamin D signaling and vitamin D-mediated anti-tumor activity in RCC, we analyzed 4 human renal cell carcinoma cell lines (Caki-1, ACHN, A498 and 786-O) and 25 samples of human RCC tumor tissue and 13 benign kidney tissues (including 10 matched tumor and benign tissue samples) by qRT-PCR, immunohistochemistry (IHC), Western blot analysis, MTT and clonogenic assays. VDR mRNA and protein were detected in all RCC cell lines. IHC showed that VDR was strongly expressed in the normal kidney tissues, but decreased in RCC tissues. As expected, VDR expression was significantly increased by treatment with 1,25-dihydroxyvitamin D3 (1,25D3) in RCC cell lines. MTT and clonogenic assays showed that 1,25D3 inhibited RCC cell growth (p Citation Format: Wei Luo, Yingyu Ma, Carmen Baldino, Justin Caserta, Swathi Ramakrishnan, Pili Roberto, Candace Johnson, Donald Trump. PIM-1 kinase regulates vitamin D receptor signaling in human renal carcinoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5056. doi:10.1158/1538-7445.AM2015-5056

Introduction: The eastern woodchuck is an established model of human hepatitis B viral infection and spontaneously develops HCC in the context of chronic woodchuck hepatitis viral infection (WHV) at a median age of 24 months. In this translational model of angiogenesis driven HCC, we evaluated the impact of sorafenib on delaying HCC development. Methods: Woodchucks were bred and inoculated at birth with titered infectious WHV pools obtained from chronic WHV carriers. By 12 months of age, the rate of chronic WHV infection was >60%. Animals were trained to receive drug orally mixed in a liquid diet. After an initial single dose woodchuck PK study it was predicted that sorafenib given orally M-F at 2.5 mg/kg once daily or 5mg/kg daily would achieve exposure in woodchucks ∼ 2x IC50 and 5X IC50 for all targets of sorafenib. This is within the exposure achieved in humans where the starting dose achieves exposures that are ∼ 10-12X IC50. We hypothesized that for both long term tolerability and delaying of HCC development, lower doses maybe ideal. Viral titers of WHV DNA were serially assessed, PK exposure of sorafenib and serial ultrasound (USG) was done q2 weeks to assess HCC development. Six WHV+ animals of similar age per arm were randomized to Placebo (P), low or high dose sorafenib. Time to tumor development (TTD) was measured from start of therapy to first occurrence of at least one 2 cm HCC was confirmed on 2 serial USGs. Tumor volume of all tumors seen was assessed as tumor burden. Results: Median TTD was 393 days, 490 and 498 days in the P, low and high dose sorafenib groups respectively. No toxicity was seen and PK results showed exposures as predicted in the two sorafenib groups. The tumor burden adjusted for the duration of therapy was significantly lower in the low dose group than the high dose and P groups. We evaluated three possible mechansims for this delay in HCC. WHV titers decreased as tumor burden increased as viral replication halts when malignant transformation occurs. The delay in TTD was not secondary to an antiviral effect that is known in this model and in humans to delay HCC occurrence. Anti-angiogenic effect was measured on H/E following necropsy as tumor necrosis and involved 7% of the total tumor volume in low dose and 13% of total tumor volume in high dose animals and 0% in P animals. High dose sorafenib inhibited the ex-vivo mixed lymphocyte reaction of woodchuck PBMC whereas low dose sorafenib did not which mechanistically may explain the difference seen in tumor burden with low compared to high dose sorafenib. Conclusions: The delay in HCC by 100 days seen in woodchucks would translate into a 3-9 years delay in HCC occurrence in humans. The tolerability at low dose for over 2 years, lower tumor burden and differential immune effects seen at low dose vs higher dose have great translational significance in the light of the recently completed STORM trial. Support: Sorafenib: Bayer. Funding: ACS- MSRG 08-096-01-CCE Citation Format: Renuka V. Iyer, Sandra Sexton, Leslie Curtin, Gerald Fetterly, Orla Maguire, Hans Minderman, Ilia Toshkov, Bud Tennant, Alan Hutson, Donald L. Trump, Candace Johnson. Low dose sorafenib delays hepatocellular cancer (HCC) development in the woodchuck model of hepatitis B related HCC. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5144. doi:10.1158/1538-7445.AM2015-5144

Intrinsic and/or acquired resistance to cisplatin is a major obstacle in the treatment of advanced bladder cancer (BC). It is important to find markers that predict tumor responsiveness to cisplatin-based chemotherapy. P73, a p53 homologue, plays an important role in cisplatin sensitivity. TAp73, a major isoform of p73, utilizes an extrinsic promoter (P1) and is pro-apoptotic. We utilized Illumina 450K methylation arrays to interrogate over 150 BC patient samples and found 9 distinct CpGs in the P1 promoter region that were hypermethylated in tumors compared to adjacent normal tissues (p Supported by NCI grants CA067267 and CA016056. Citation Format: Rebecca D. Greenspan, Nithya Krishnan, Carl Morrison, Angela Omilian, Wiam Bshara, Amber Worral, Kristopher Attwood, Donald L. Trump, Anna Woloszynska-Read, Candace S. Johnson. P73, a potential marker for chemoresponsiveness to cisplatin therapy and survival in muscle invasive bladder cancer (MIBC). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-158. doi:10.1158/1538-7445.AM2015-LB-158

Vitamin D is a steroid hormone that regulates calcium homeostasis and bone metabolism. The active form of vitamin D [1α,25-dihydroxyvitamin D3 (1,25D3)] acts through both genomic and nongenomic pathways. 1,25D3 has antitumor effects in a variety of cancers, including colorectal, prostate, breast, ovarian, and skin cancers. 1,25D3 exerts growth-inhibitory effects in cancer cells through the induction of apoptosis, cell cycle arrest, and differentiation. The mechanisms regulating 1,25D3-induced apoptosis remain unclear. We investigated the role of nongenomic signaling in 1,25D3-mediated apoptosis in squamous cell carcinoma (SCC) cells. 1,25D3 induced rapid and sustained activation of phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) 1/2 pathways in SCC cells. These effects were nongenomic: they occurred rapidly and were not inhibited by cycloheximide or actinomycin D. To examine whether the nongenomic activation of Akt and ERK1/2 plays a role in 1,25D3-mediated apoptosis, the expression of Akt or ERK1/2 was reduced by small interfering RNA (siRNA). siRNA-Akt significantly enhanced 1,25D3-induced apoptosis as indicated by increased levels of Annexin V–positive cells and increased sub-G1 population and DNA fragmentation. In contrast, siRNA-ERK1/2 had no effects on 1,25D3-induced apoptosis. In addition, siRNA-Akt transfection followed by 1,25D3 treatment induced apoptosis much sooner than 1,25D3 alone. siRNA-Akt and 1,25D3 induced caspase-10 activation, suppressed the expression of c-IAP1 and XIAP, and promoted 1,25D3-induced caspase-3 activation. These results support a link between 1,25D3-induced nongenomic signaling and apoptosis. 1,25D3 induces the activation of phosphatidylinositol 3-kinase/Akt, which suppresses 1,25D3-mediated apoptosis and prolongs the survival of SCC cells. (Cancer Res 2006; 66(16): 8131-8)

Progress has recently been made in identifying populations at risk for lung cancer using genetic, clinical and demographic information. The N-nitroso-tris-chloroethylurea (NTCU) mouse model, in which animals develop premalignant histopathology similar to that seen in humans, is used to examine the potential efficacy of chemoprevention agents to be utilized in at risk populations. We identified 25 µl of 40 mM NTCU /week as the optimal dosing regimen in SWR/J mice for chemoprevention studies. At this dose, topical treatments of NTCU induce predominantly low-grade dysplastic lesions by 15 weeks (w) and high-grade dysplastic (HGD) lesions by 25 w in the large airways. Additionally we found that NTCU stimulates a state of chronic inflammation associated with the development of dysplasias. Epidemiologic studies indicate that there is an inverse relationship between vitamin D and the risk and prognosis of lung cancer. Vitamin D acts through the vitamin D receptor to promote cellular differentiation and inhibit proliferation and inflammation. The effect of vitamin D on cancer progression was tested in the NTCU model, using dietary vitamin D3 (0 or 2000 IU/kg) alone and in combination with intraperitoneal injections of the active metabolite of vitamin D, calcitriol (80 ug/kg). Female mice were randomized to 6 treatment groups (n=15 mice/group/time point (15 and 25 w)). Disease was evaluated by enumerating the percentage of HGD lesions, per the total area in serial H&E sections of the lung. The percentage of HGD lesions in the large airways was reduced in the vitamin D sufficient mice (SN) (8.72%, P Furthermore, there was a significant increase in proliferation associated with increased HGD. Following 25 w of NTCU treatment there was a 2-fold increase in Ki-67 staining all groups compared all groups after 15 w of NTCU. However, the was 30% more Ki-67staining in the DN (P Citation Format: Sarah A. Mazzilli, Mary E. Reid, Paul N. Bogner, Kristopher Attwood, Pamela A. Hershberger, Donald L. Trump, Candace S. Johnson. Vitamin D sufficiency slows the progression of dysplasic lesions in the NTCU mouse model of lung squamous cell carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 238. doi:10.1158/1538-7445.AM2014-238

Introduction: Better animal models that recapitulate the liver milieu of human HCC are needed. The eastern woodchuck is an established model of human hepatitis B viral infection and spontaneously develops HCC in the context of chronic woodchuck hepatitis viral infection (WHV). The translational relevance of this model for developing anti-angiogenic therapies was evaluated using sunitinib (S), a potent oral, anti-angiogenic agent. Methods: Woodchucks were bred and inoculated at birth with titered infectious WHV pools obtained from chronic WHV carriers. By 12 months of age, the rate of chronic WHV infection was >60%. Carriers were followed by USG, upon developing HCC, 12 animals were randomized 1:1 to S or placebo (P) given once orally daily for 30 days. From a single treatment S PK study at 4 dose levels, n=3/group, simulations showed 12mg/kg daily was expected to be optimal for achieving steady state serum concentrations between 50- 100 ng/ml in woodchucks. Tumor size and blood flow were assessed using dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) before treatment and on day 28 using standardized protocols. At study completion or when animals were humanely euthanized, tumors and any other small nodules were fixed overnight in 10% buffered formalin. After standard processing and embedding in paraffin, 4 μm sections were prepared, deparaffinized, stained with hematoxylin-eosin (H&E), studied with a Zeiss Axio Imager A1 microscope by a pathologist blinded to the treatment arm. Morphometric study of areas of necrosis were compared to the size of the tumor sections by counting view fields at medium objective magnification, and differences in percentages between the groups were compared by the Excel student's two tailed, two-sample unequal variance t-test. Results: The median age was 30 months. Four animals died during the course of the study (1P, 3 S) and were replaced. Median therapy duration was 28 days. Morphologically, portal hepatitis and pre-neoplastic lesions (foci of altered hepatocytes [FAH]) and larger areas of altered hepatocytes (AAH) were seen in all livers. Areas of tumor necrosis of varying size (20-100%) adjacent to terminal hepatic venules seen in both high grade and in rare, low grade tumor sections of S treated woodchucks were rare in P animals. The difference was highly significant (p= 6.0 E-13) between the two groups. By DCE-MRI, median tumor volume change was + 17% and -13% and necrotic tumor volume change was +22% and +190% in P and S treated animals respectively. However all MRI parameters (k trans, AUC90, median tumor volume) were not statistically significantly different between the groups. Conclusions: Angiogenic response across different stages of hepatocarcinogenesis can be studied in woodchucks. Histological necrosis in S treated woodchucks without significant DCE-MRI change and the toxicities seen with S have translational significance to human HCC. Citation Format: Alexander Pomakov, Ilia Toshkov, Sandra Buitrago, Leslie Curtin, Donald Trump, Candace Johnson, Edward Ashton, Bud Tennant, Renuka Iyer. Developing anti-angiogenic therapies for human hepatocellular cancer (HCC)- studies of suntinib in the woodchuck model of hepatitis B related HCC. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4946. doi:10.1158/1538-7445.AM2014-4946

Bladder cancer (BC), 90% of which have transitional cell histology, is the fourth most common malignancy in the US. Transitional cell carcinoma (TCC), which occurs predominantly in men and whose incidence appears to be increasing, occurs most frequently as superficial disease which, while prone to recurrence, is not life threatening. However, TCC which invades the muscle layer of the bladder (stage T2-T4) is lethal in ∼50% of individuals. Stage T2-T4 TCC is present at initial diagnosis in ∼30% of patients and superficial TCC progresses to T2-T4 disease 20% of the time. We sequenced whole exomes of 28 muscle-invasive bladder tumors and performed an additional screen of over 200 genes in 119 additional TCCs. We found frequent alterations (10%) in STAG2, a gene encoding a subunit of the cohesin complex, which regulates the sister chromatids segregation process. Most mutations identified on STAG2 were nonsense, missense or frameshift. Genetic disruption of the sister chromatid segregation process leads to aneuploidy, which is one of the hallmarks of cancer. Based on recently published studies, however, it is uncertain whether a loss of STAG2 causes aneuploidy or not. In this study we sought to elucidate the role of STAG2 loss of expression in bladder tumorigenesis. We have analyzed the expression of STAG2 in 14 urothelial cell lines at both mRNA and protein levels by means of q-PCR and western blots, respectively. We found that STAG2 expression was lost in 2 of the 14 urothelial cell lines (HB-CLS-2 and UM-UC-3) analyzed. We chose SV-HUC (immortalized normal urothelium cells) along with two bladder cancer cell lines that had high expression of STAG2 (HB-CLS-1 and TCC-SUP) to study effects of STAG2 loss by transient knockdown. Knockdown of STAG2 in SV-HUC, TCC-SUP and HB-CLS-1 cells did not affect proliferation. Cell cycle analysis on TCC-SUP cells revealed that STAG2 knockdown resulted in a lower percentage of cells in the G0/G1 phase in comparison to the scrambled control. Taken together, these data suggest that STAG2 has a role in modifying DNA content but not proliferation. Our ongoing studies utilizing bladder cancer tissue microarray (TMAs) and copy number variation (CNV) array will help to clarify the consequences of STAG2 loss in bladder cancer. Citation Format: Nithya Krishnan, Anna Woloszynska-Read, Jianmin Wang, Song Liu, Carl Morrison, Khurshid Guru, Evelyn Smit, Donald Trump, Candace Johnson. Loss of STAG2 in bladder cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2226. doi:10.1158/1538-7445.AM2014-2226

Bladder cancer is the 5th most common in the United States. 30% of cases are advanced stage requiring multimodality therapy at diagnosis and many which are first diagnosed when superficial will progress to more advanced disease. High recurrence and progression rate and drug resistance highlight the need for new therapeutic strategies to improve clinical outcomes. 1,25-dihydroxyvitamin D3 (1,25D3), the most active vitamin D metabolite, has demonstrated broad spectrum antitumor activities in vitro and in vivo. In addition, 1,25D3 potentiates the antitumor effects of common chemotherapeutic agents in several cancer cell types including bladder cancer. 24-hydroxylase, encoded by the CYP24A1 gene, plays a key role in degrading 1,25D3. Increased expression of CYP24A1 has been found in several human tumors. Several studies reveal that the protein kinase signaling pathways including PIM1 are involved in the regulation of CYP24A1 expression. The PIM kinases are a family of serine/threonine kinases (PIM-1, PIM-2, PIM-3) that have been associated with tumorigenesis and drug resistance. PIM1 has been extensively studied in lymphoma and upregulation of PIM kinase expression has been reported in many malignancies. We analyzed mRNA expression of CYP24A1 and PIM1 in 17 bladder cancer cell lines by qRT-PCR. We found that CYP24A1 expression is increased in 12 out of 17 bladder cancer cell lines and PIM1 expression is increased in 8 out of 17 bladder cancer cell lines compared to expression in a benign bladder cancer cell line. Therefore, we hypothesized that inhibition of PIM kinase activity would suppress CYP24A1 expression and thus enhancing 1,25D3-mediated anti-tumor activity. Using the PIM1 inhibitor, JP11646, we found that inhibition of PIM1 kinase activity reduces CYP24A1 expression at transcriptional level in bladder cancer cells RT112 and RT112D21 which express high level of PIM1 and CYP24A1. We further evaluated the efficacy of the PIM1 inhibitor alone and in combination with 1,25D3. Inhibition of PIM1 kinase activity by JP11646 reduces cell growth in a panel of bladder cancer cell lines in a dose-dependent manner as measured by MTT assays. MTT assays detected a significant inhibition of cell proliferation by 50% compared to the control when cells were treated with 128 nM of JP11646 for 72 h in 6 of bladder cancer cell lines tested. Furthermore, inhibition of PIM1 kinase activity enhances 1,25D3-mediated inhibitory effect on cell growth in bladder cancer cells RT112 and RT112D21. This study provides the first evidence that inhibition of PIM kinase activity may be an attractive therapeutic strategy to facilitate the optimization of antitumor therapy of vitamin D in bladder cancer. This study was supported by NIH/NCI grants 5R01CA067267 and 5R01CA095045 Citation Format: Wei Luo, Carmen M. Baldino, Justin Caserta, Yingyu Ma, Candace Johnson, Donald L. Trump. Targeting PIM1 kinase enhances 1,25-dihydroxyvitamin D3-mediated anti-tumor activity in bladder cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 607. doi:10.1158/1538-7445.AM2014-607

Prostate-specific antigen (PSA) is currently used as a biomarker for the early diagnosis and management of prostate cancer (CaP). However, PSA generally lacks the sensitivity and specificity desired of an effective diagnostic biomarker. The goal of this study was to identify an additional biomarker or a panel of biomarkers that is more sensitive and specific than PSA in differentiating benign vs. malignant prostate disease and/or localized CaP vs. metastatic CaP. Concurrent measurements of circulating IL-8, TNF-alpha (TNF-α) and sTNFR1 were obtained from four groups of men: (1) controls (2) men with elevated PSA with a negative prostate biopsy (eIPSA_negBx) (3) men with clinically localized CaP and (4) men with castration resistant CaP (CRPC). TNF-α (AUC=0.93) and sTNFR1 (AUC=0.97) were strong predictors of elPSA_negBx vs CaP). The best predictor of elPSA_negBx vs prostate cancer was sTNFR1 and IL8 combined (AUC=0.997). The strongest single predictors of localized versus metastatic CaP were TNF-α (AUC=0.992) and PSA (AUC=0.963) levels. Conclusions: The specificity and sensitivity of a PSA-based CaP diagnosis can be significantly enhanced by concurrent serum measurements of IL-8, TNF-α and sTNFR1. In view of the concerns about the ability of PSA to distinguish clinically relevant CaP from indolent disease, assessment of these biomarkers in larger cohort consisting of Caucasians and African Americans is warranted. Citation Format: Kailash C. Chadha, Austin Miller, Bindukumar B. Nair, Stanley A. Schwartz, Donald L. Trump, Willie Underwood. New serum biomarkers for prostate cancer diagnosis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 879. doi:10.1158/1538-7445.AM2014-879

In a murine squamous cell carcinoma (SCC) model, we have demonstrated that both 1,25-dihydroxycholecalciferol (1,25-D3) and the analogue 1,25-dihydroxy-16-ene-23-yne-cholecalciferol (Ro23-7553) have significant in vitro and in vivo antitumor activity. We have examined here the cell cycle effect of 1,25-D3 and Ro23-7553 on SCCVII/SF tumor cells by quantitating nuclear DNA using a detergent-trypsin method via flow cytometry analysis. Both 1,25-D3 and Ro23-7553 resulted in a significant increase of cells in G0-G1, with an accompanying decrease of cells in S phase. The ability to arrest cells in G0-G1 has been exploited by combining Ro23-7553 with the cytotoxic agent cisplatin (cis-diamminodichloroplatinum; cDDP). Using the in vitro clonogenic assay, pretreatment with Ro23-7553 for 24-48 h significantly enhanced cDDP-mediated tumor cell kill as compared to concurrent treatment with Ro23-7553 and cDDP or cDDP alone. To examine the effect of Ro23-7553 and cDDP in vivo, C3H/HeJ mice with 9-14-day SCC tumors were treated either for 3 days with varying i.p. doses of Ro23-7553 or for 7 days continuously through the use of Alzet pumps, and on the last day of Ro23-7553 treatment, cDDP (1-6 mg/kg) was administered. Using the in vivo excision tumor cell clonogenic assay, in which tumors were removed from animals 24 h after cDDP treatment and plated in a clonogenic assay, pretreatment with Ro23-7553 markedly enhanced cDDP-mediated clonogenic tumor cell kill, even at low doses of cDDP as compared to cDDP treatment alone. Similarly, a significant decrease in fractional tumor volume and increase in tumor regrowth delay was observed when animals were pretreated before cDDP with Ro23-7553 as compared to either agent alone. These results demonstrate a significant enhanced antitumor effect with Ro23-7553 pretreatment before cDDP both in vitro and in vivo and suggest that Ro23-7553 may potentiate cDDP cytotoxicity through effects on cell cycle progression.

Epidemiological data suggest an important role of vitamin D signaling in cancer progression, and experimental studies demonstrate that the active vitamin D metabolite 1α, 25-dihydroxyvitamin D3 (1,25D3) has broad spectrum anti-tumor activity. Hypercalcemia has often been suggested to limit the clinical application of these data. The 14-epi-analog of 1,25D3, inecalcitol (19-nor-14-epi-23-yne-1,25-(OH)2D3; TX522), was developed to have superagonistic anti-tumor activities but low hypercalcemia potential. We examined the anti-tumor activity of inecalcitol and the underlying mechanisms in a murine squamous cell carcinoma (SCC) model system. Compared to 1,25D3, inecalcitol showed enhanced vitamin D receptor (VDR)-mediated transcriptional activity. Inecalcitol suppressed SCC cell proliferation in a dose dependent manner with an IC50 value 30 times lower than that of 1,25D3. Both inecalcitol and 1,25D3 induced a comparable level of G0/G1 cell cycle arrest in SCC cells. The level of apoptosis induced by inecalcitol was markedly higher than that of 1,25D3. Apoptosis was mediated through the activation of the caspase 8/10- caspase 3 pathway. Further, inecalcitol markedly inhibited the mRNA and protein expression of c-IAP1 and XIAP compared to 1,25D3. In vivo, inecalcitol inhibits SCC tumor growth at 80 μg dose without hypercalcemia. Inecalcitol (320 μg) or 1,25D3 (0.312 μg) markedly inhibited tumor growth. Notably, inecalcitol induced a significantly higher level of apoptosis in the SCC xenograft model. We show that inecalcitol has potent anti-tumor activity in the SCC model system and this is associated with a stronger induction of apoptosis. These findings support the further development of inecalcitol in cancer treatment. Citation Format: Yingyu Ma, Wei-Dong Yu, Alejandro A. Hidalgo, Wei Luo, Remi Delansorne, Candace S. Johnson, Donald L. Trump. Inecalcitol, an analogue of 1,25D3, displays enhanced antitumor activity through the induction of apoptosis in a squamous cell carcinoma model system. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3434. doi:10.1158/1538-7445.AM2013-3434

The traditional role of vitamin D is in the control of calcium homeostasis and bone mineralization. Over the past 2 decades, studies have demonstrated that vitamin D is useful for the prevention and treatment of various cancers. 24-hydroxylase, encoded by CYP24A1 gene, is the key enzyme which inactivates vitamin D. Increased expression of CYP24A1 has been found in several human tumors. CYP24A1 is regulated directly by 1,25(OH)2D3 (1,25D3) through genomic signaling. Studies have revealed that protein kinase signaling pathways may also be involved in the regulation of CYP24A1 expression. By screening a chemical compound library, we discovered that small chemical compounds with inhibitory activity toward protein kinases including PIM1 and HIPK2 inhibited CYP24A1 expression. Furthermore, siRNA-mediated PIM1 knockdown reduced 1,25D3-induced CYP24A1 mRNA and protein expression in human prostate cancer PC3 cells. In contrast, HIPK2 knockdown had no effect. The serine/threonine kinase PIM1 has been implicated as an oncogene in various human cancers including lymphomas, gastric, colorectal and prostate carcinomas. We analyzed mRNA expression of CYP24A1 and PIM1 in 26 case-matched pairs of human benign and malignant prostate by qRT-PCR. CYP24A1 expression was increased in 6 out of 26 malignant prostate samples compared to benign lesions (> 1.5 fold). PIM1 expression was increased in 10 out of 26 malignant prostate samples compared to benign lesions (> 1.5 fold). Five of the 6 samples with increased CYP24A1 in malignant lesions also had increased PIM1 expression. Fisher's exact test shows that increased CYP24A1 expression is significantly associated with increased PIM1 expression in malignant prostate (P = 0.0184). These data indicate that PIM1 may be involved in increased CYP24A1 expression in prostate cancer. This study was supported by NIH/NCI grants 5R01CA067267 and 5R01CA095045. Citation Format: Wei Luo, Mikhail Chernov, Yingyu Ma, Candace Johnson, Donald Trump. Over-expression of PIM1 kinase contributes to CYP24A1 gene up-regulation in prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1819. doi:10.1158/1538-7445.AM2013-1819

Vitamin D, an essential mediator of calcium homeostasis, acts through the vitamin D receptor to promote cellular differentiation and inhibit proliferation. Epidemiologic studies indicate that low serum levels of 25(OH)D3 are associated with increased risk and poor prognosis of lung cancer. Pre-clinical studies in mice show that dietary vitamin D and calcium deficiency promote carcinogenesis in mouse models of cancer. These data suggest that vitamin D may have a role as a chemopreventive agent. To investigate the role of vitamin D in lung cancer, a carcinogen-induced model of lung squamous cell carcinoma (SCC) was employed. N-nitroso-tris-chloroethylurea (NTCU) was applied topically each week to the back of SWR/J mice. Following ∼15 weeks of NTCU treatment, mice on a standard diet begin to develop flat atypia, which progresses through dysplasia to invasive lung SCC by 35 to 40 weeks. We investigated the effects of vitamin D on cancer progression using different levels of dietary vitamin D3 and injections of the most active metabolite 1,25(OH)2D3 (calcitriol) in the NTCU model (80 mM/week). After 24 weeks of NTCU, mice on a deficient diet had more advanced disease than those on a sufficient diet. However, that study demonstrated that the 80 mM/week of NTCU was too toxic and resulted in premature death. A second vitamin D intervention study has now been completed using a less toxic dose of 40 mM/week of NTCU. In this study mice were randomized to 6 treatment groups (Table 1). Disease burden, as measured by histology (Table 1), was similar to that seen with higher dose NCTU. This study supports our earlier findings that vitamin D deficient mice develop disease at a more rapid rate when compared to mice that are sufficient. This also suggests that calcitriol administration may reduce the progression of lung SCC in mice that are vitamin D deficient. Further studies are on going to elucidate the mechanism by which vitamin D acts to slow the progression of lung SCC in this model. Table 1. Serum 25(OH)D3 levels & incidence of high-grade dysplasia and lung SCC. Group Carcinogen (NTCU) Diet Vitamin D (IU/kg) Serum 25(OH)D3 Level (ng/ml) High-grade Dysplasia (%) Lung SCC (%) D deficient − 0 Citation Format: Sarah A. Mazzilli, Mary Reid, Paul Bogner, Donald Trump, Candace Johnson. Vitamin D reduces lung cancer progression the N-nitroso-tris-chloroethylurea model of mouse lung squamous cell carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 178. doi:10.1158/1538-7445.AM2013-178

Background: Experimental and ecological studies support links between vitamin D and prostate cancer prevention and prognosis. Given the lower levels of circulating 25-hydroxyvitamin D [25(OH)D] and higher prostate cancer incidence and mortality among African Americans compared to other racial/ethnic groups, the aim of this investigation was to examine the relationship between plasma 25(OH)D levels and prostate cancer aggressiveness among African and European Americans. Methods: Plasma 25(OH)D was measured using LC/MS/MS in 537 African Americans and 663 European Americans with newly-diagnosed prostate cancer from PCaP. Men were classified as cases (high aggressiveness) if Gleason sum ≥8, or PSA >20 ng/ml, or Gleason sum ≥7 AND clinical stage = T3c-T4c, or Gleason sum=7 with primary pattern 4. The comparison group (low aggressiveness) included men with Gleason sum Results: African Americans had significantly lower mean concentrations of 25(OH)D (17.7 ± 7.6 ng/ml) compared to European Americans (24.6 ± 9.6 ng/ml). The highest tertile (mean=25.79 ng/ml & median=24.29 ng/ml) and middle tertile (mean=16.09 ng/ml & median=15.83) of plasma 25(OH)D were positively associated with highly aggressive prostate cancer among African Americans after adjustment for age, season, education, physical activity, smoking status, and PSA screening history (OR=1.7, 95% CI=1.0, 2.8 and OR=1.8, 95%CI=1.1, 3.0, respectively). No significant associations were observed for European American men. Conclusions: Plasma 25(OH)D was positively associated with prostate cancer aggressiveness among African Americans but not European Americans, such that subjects with highly aggressive prostate cancer had increased odds of having higher plasma 25(OH)D levels. Research samples were collected after diagnosis, so effects of treatment, extent of disease or associated processes (e.g. weight loss) on plasma 25(OH)D may explain the findings. Ongoing studies include analysis of vitamin D binding protein (DBP) in the plasma and genotyping of DBP affinity variants in PCaP subjects. This approach may help explain the racial differences found, since DBP may modulate the impact of vitamin D status on prostate cancer. Citation Format: Anna Woloszynska-Read, Lenore Arab, John Adams, Jeannette T. Bensen, Elizabeth TH Fontham, James L. Mohler, Joseph Su, Fred Tabung, Hongmei Zhang, Donald L. Trump, Candace S. Johnson, Susan E. Steck. Plasma 25-hydroxyvitamin D levels are associated with aggressive prostate cancer among African Americans in the North Carolina-Louisiana Prostate Cancer Project (PCaP). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-12. doi:10.1158/1538-7445.AM2013-LB-12

Lung cancer accounts for the majority of cancer mortality in both men and women. It is critical to better understand the biology of lung cancer development as no preventive agents and few effective treatment options exist for lung cancer. Epidemiological studies indicate that low serum levels of 25(OH)D3 are associated with increased risk and poor prognosis of lung cancer. Calcitriol, the active metabolite of vitamin D, induces cell cycle arrest, apoptosis and differentiation in preclinical and clinical studies, has limited toxicity, and hence, is an attractive agent for chemoprevention. To investigate the preventive effects of vitamin D on lung squamous cell carcinoma (SCC), pre-clinical studies were conducted using a carcinogen-induced (CI) mouse model of lung SCC which uses N-nitroso-tris-chloroethylurea (NTCU) applied topically to the back of a SWR/J mouse twice weekly. Following ∼15 weeks of NTCU treatment, mice begin to develop bronchial epithelial hyperplasia and in time, progress through metaplasia, carcinoma in situ, and ultimately form lung SCC by approximately 35 to 40 weeks. We conducted a preliminary study investigating the effects of vitamin D on cancer progression in the CI model where animals received NTCU (80 mM/week). 12 week old female mice were randomized into one of three treatment groups: 1) vitamin D deficient diet (DD) (0IU D3 in the diet), 2) sufficient diet (SD) (2000IU/day D3 in the diet) and 3) SD with weekly intraperitoneal (IP) injections of calcitriol (80 ug/kg) (SD+D). Animals treated with NTCU on a DD, SD and SD+D had average serum 25(OH)D3 levels of 15, 70 and 75 nmol/L, respectively (normal range in mice is 70- 90 nmol/L). Preliminary histological evaluation at 12 weeks revealed bronchial epithelial hyperplasia in both the SD and SD+D groups. There were more advanced changes including hyperplasia, metaplasia and dysplasia in the DD mice. The histological evaluation of the DD mice at 24 weeks revealed even more advanced disease, in that most mice had developed lung SCC. The histological evaluation of the SD and SD+D groups at 24 weeks indicated the presence of bronchial epithelial hyperplasia, squamous metaplasia and dysplasia in all mice with some low level of carcinoma in situ. This study indicates that mice deficient in vitamin D develop disease at a more rapid rate suggesting that vitamin D status may play an important role in progression of lung SCC. Supported by NCCAM F31 AT006487 & R01-CA-067267. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1606. doi:1538-7445.AM2012-1606

Progression risk from early stage localized prostate cancer (CaP) to more aggressive and lethal phenotypes is not precisely predicted. This leads to the over-treatment of men whose disease might otherwise remain indolent. Tumor and serum microRNA (miR) expression show promise as functional biomarkers of CaP progression risk but considerable ambiguity exists over which miR are altered, in which direction and at what point in disease. To address these ambiguities we undertook a genome-wide miR expression comparison using a micro-array approach in key and isogenic CaP cell lines and non-malignant controls. Specifically, cell pairs that represent; Initiation (non-malignant RWPE-1 and RAS-transformed RWPE-2 cells); Progression (non-malignant HPr-1AR and LNCaP cells); Recurrence (LNCaP and LNCaP-C4-2 cells). Comparison between these cell line pairs identified 56 altered miR (≥1.5 fold, p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-477. doi:1538-7445.AM2012-LB-477

Prostate cancer exhibits striking racial disparity; African American (AA) men have a markedly higher risk of being diagnosed and of dying of prostate cancer compared to Caucasian American (CA) men. This disparity may be due to biologic differences associated with race or to sociologic factors, such as economic disparities or differences in access to care or interactions of all these factors. Differences in tumor gene expression and methylation might be associated with the poorer prognosis seen among AA men and may contribute to better understanding of these disparities and the delineation of new therapeutic targets. In order to study the effect of the tumor microenvironment on endothelial cells found in tissue from AA and CA patients with prostate cancer, we first established a method for the isolation of the endothelial cells from malignant and benign prostate tissue obtained at prostatectomy. We then conducted a pilot study of transcriptome and methylome analysis in 5 pairs of CD31+ endothelial cells isolated from paired samples of benign and malignant tissue from AA and CA patients with prostate cancer. We have identified 2,096 and 920 differentially expressed transcripts for the tumor versus normal in AA and CA group, respectively. However, only 140 transcripts are shared. Furthermore, a total of 2,248 transcripts show different level of expression change for tumor versus normal in samples with AA origin compared to samples with CA origin. Genome-wide DNA methylation profiling in the same endothelial samples have identified 18,093 differentially methylated loci in AA group, and 12,372 differentially methylated loci in CA group. Only a total of 1,324 loci are shared. Remarkably, there are a total of 20,164 loci with different level of methylation change for tumor versus normal in AA group compared to CA group. Integrative analysis of transcriptome and methylatome data showed that a large percentage of differentially expressed genes have loci whose methylation change is inversely correlated with gene expression change, and vice versa. Taken together, our pilot data, while preliminary, suggest the possibility that there is a potential racial difference in terms of differential expression and methylation between CA and AA endothelial cell populations. Future large studies are warranted to confirm our findings and further explore the role of race disparity in the delineation of novel therapeutic targets to be utilized clinically for prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4280. doi:1538-7445.AM2012-4280

The pharmacokinetics of cyclophosphamide (CP) and several important metabolites was studied in detail in six patients receiving CP alone and with a radio- and chemosensitizing agent, SR-2508. CP at 1000 mg/m2 was either infused in 20 min alone or given 2 h before an infusion of SR-2508 at 5 g/m2 over 20 min, both separated by 3 weeks, to the same patients in a randomized fashion. Plasma and 24-h urinary levels of CP and four metabolites: [4-hydroxycyclophosphamide (4-OH CP), phosphoramide mustard (PM), chloroethyl oxazolidin-2-one, and alcophosphamide] were monitored by a gas chromatographic-mass spectrometric-stable isotope dilution assay. CP plasma levels were found to decline monoexponentially with the appearance of transient saturation kinetics in some and a mean t1/2 of 5.2 h for patients treated with CP alone. Plasma 4-OH CP levels showed a mean peak concentration of 2.4 microM and declined approximately in parallel to those of CP. The major circulating metabolite was found to be PM with a mean peak concentration of 40 microM and a terminal t1/2 of 15 h. The mean area under the plasma concentration curve (AUC) ratios between metabolites and CP were: 4-OH CP, 0.0158; PM, 0.4518; and chloroethyl oxazolidin-2-one, 0.179 with alcophosphamide at low levels. No appreciable amount of nornitrogen mustard was detected. Mean urinary excretion was: CP, 10.8; 4-OH, CP, 0.5; PM, 39.0; alcophosphamide, 0.4; and chloroethyl oxazolidin-2-one, 3.0, all expressed as a percentage of CP dose. No statistically significant difference was detected in all standard pharmacokinetic parameters determined for both CP and metabolites between patients with CP alone and with SR 2508. Plasma 4-(p-nitrobenzyl)pyridine activity was found to correlate the closest with PM profiles, with respect to both standard pharmacokinetic parameters and AUC values. When plasma PM AUC values were plotted against AUC values of circulating 4-(p-nitrobenzyl)pyridine activity, a correlation coefficient of 0.859 (P0.001) was obtained. Together with the significant cytotoxicity of PM these data support a significant contribution of circulating PM in the antitumor effect of PM.