Citations

Abstract BACKGROUND: Biobanks collect and store biological samples and data from participants for use in research by multiple investigators. The Mayo Clinic Biobank was established by the Mayo Clinic Center for Individualized Medicine with a goal to support a wide array of health-related research studies at Mayo Clinic, including cancer studies. The Biobank is not focused on any particular disease, however, through annotation by questionnaires, the Mayo electronic medical record (EMR), and the Mayo Cancer Registry, it is a useful clinical research resource for cancer related studies. METHODS: Recruitment started in April 2009. Our goal is 50,000 enrollees. Eligible subjects are adult Mayo Clinic patients, U.S residents, and able to consent. Mailed invitations were sent to patients with appointments in certain Mayo Clinic departments. Each participant completed a health history questionnaire including a wide variety of health information as well as whether they or their 1st-degree relatives had ever been diagnosed with specific cancers. For self-reported cancers, subjects were asked to provide the age they were first diagnosed. Subjects also provided a blood sample and granted research access to clinically collected specimens and data from their Mayo Clinic record, at enrollment and into the future. All research projects utilizing samples from this resource are required to return to the Biobank the data generated from their projects. RESULTS: In the first three years, 21736 subjects consented to participate (30% response rate). Participants are 58% female, 95% White, and range in age from 18 to 97 with a median age of 62. At enrollment, 74% were residents in Minnesota, 6% in Iowa, 4% in Wisconsin, and 15% in the other states. Twelve percent reported being a current smoker, while 59% were never smokers. The most common self-reported cancers at baseline were non-melanoma skin cancer (n=2950), prostate cancer (n=1107), breast cancer (n=941), melanoma (n=692), colorectal cancer (n=296), cervical cancer (n=240), sarcoma (n=233), urinary/bladder (n=211), lymphoma (n=187), thyroid cancer (n=186), endometrial cancer (n=170), kidney cancer (n=163), lung cancer (n=145). Excluding non-melanoma skin cancer, 16188 (74%) (6516 male, 9672 female) self-reported being cancer free at baseline. Data will be presented on the frequency of incident cancers diagnosed since enrollment in the Biobank. To date, the Biobank has served as a source of controls for 17 cancer related studies on genetic susceptibility, including studies of the breast, colon, myeloma, brain, lung, pancreas, endometrium, and kidneys. CONCLUSIONS: The Mayo Clinic Biobank has quickly been established as a valuable resource for cancer researchers at Mayo Clinic - primarily as a source of controls for genetic studies. As the Biobank continues to grow in sample size and length of follow-up, it will also serve as a cohort study of cancer incidence embedded within an EMR of a large health organization. Citation Format: Janet E. Olson, Euijung Ryu, Kiley J. Johnson, Karen Maschke, Jody A. Morrisette, Mark Liebow, Paul Y. Takahashi, Ruchi G. Sharma, Kari S. Anderson, Matthew A. Hathcock, Jyotishman Pathak, Noralane M. Lindor, Timothy J. Beebe, Stephen N. Thibodeau, James R. Cerhan. The Mayo Clinic Biobank - A growing resource for cancer related studies. [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 2515. doi:10.1158/1538-7445.AM2013-2515

Abstract NSC 373981 is 1-ethanol, 2-nitro-naphtho[2,1-b]furan that has demonstrated activity in the NCI60 anticancer screen and in the mouse hollow-fiber assay. Growth inhibitory activity was also observed in several xenograft models. There is some evidence that antitumor activity is greater in vivo than would be predicted by the in vitro results, suggesting the possibility of activating metabolism. While the mechanism of anticancer activity is unknown, naphthofurans have been reported to modulate certain nuclear receptors. The purpose of this study was to characterize the plasma pharmacokinetics of NSC 373981 following i.v. and i.p. administration to athymic nude mice and to characterize potential metabolites of this compound in plasma, urine, and bile. We developed and validated suitable positive ionization LC/MS/MS methodology for measuring NSC 373981 that utilized liquid extraction with 1-chlorobutane and MS/MS detection with the 258>212 and 244>169 transitions, respectively, for NSC 373981 and NSC 329226, an analog that was used as the internal standard. Baseline separations were achieved on a Polaris C18 column under a mobile phase consisting of 70:30 methanol:water containing 0.1% formic acid. Standard curves were linear over the concentration range 2.5-1000 ng/mL with a lower limit of detection of 2.5 ng/mL. NSC 373981 was stable under acidic, neutral and basic conditions, as well as human and mouse plasma during 24-hr incubation at 37°C. NSC 373981 pharmacokinetics were characterized in male nu/nu mice after a 37.5 mg/kg i.v. dose or a 150 mg/kg i.p. dose. Plasma, bile, urine and feces were collected for 24-hr after drug administration. Following i.v. administration, the peak concentration was > 5.9 μg/mL and the terminal elimination half-life was 1.2 hours. Volume of distribution and plasma clearance values were 694 ml and 390 mL/hr, respectively. Following i.p. administration, NSC373981 reached a peak concentration of 2.1 μg/mL at 15 minutes and the terminal elimination half-life was 36.8 hours. The i.p. bioavailability of NSC373981 was 58%. In vivo metabolite profiles were characterized in plasma, urine, feces, and bile. We detected several Phase I metabolites in these samples including those produced by reduction of the aromatic nitro group, oxidation of the primary alcohol moiety on the reduced metabolite, oxidation of the primary alcohol moiety, benzylic hydroxylation, hydroxylation and dihydrodiol formation on the naphthalene moiety, and Phase II metabolites including glucuronidation of the carboxylic acids and a glutathione metabolite of hydroxylated NSC 373981. Supported by NCI contract N01-CM-2011-00014. Citation Format: Joel M. Reid, Sarah A. Buhrow, Renee M. McGovern, Joseph M. Covey, Matthew M. Ames. Pharmacokinetics and in vivo metabolism of the naphthofuran NSC373981 in male nu/nu mice. [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 2462. doi:10.1158/1538-7445.AM2013-2462

Abstract The FKBP5 gene encodes the FKBP51 immunophilin, a negative regulator of AKT. Variability in level of FKBP5 expression is a major factor contributing to variation in response to several classes of chemotherapeutic agents including gemcitabine, first line treatment for pancreatic cancer. Genetic variation of FKBP5 could influence its expression, function, and, ultimately, treatment response of pancreatic cancer. We set out to comprehensively study the role of genetic variation in FKBP5 identified by Next Generation DNA resequencing in response to gemcitabine treatment of pancreatic cancer by utilizing both tumor and germline DNA samples from 43 pancreatic cancer patients, including 19 paired tumor-normal samples. Genotype-phenotype association studies with overall survival (in tumor only) as well as with FKBP5 gene expression were performed using the same samples in which Next Generation DNA resequencing had been performed, followed by functional genomic studies of top selected single nucleotide polymorphisms (SNPs). In-depth resequencing identified 404 FKBP5 SNPs in both normal and tumor DNA patient samples. SNPs with the smallest p-values for association with FKBP5 survival or expression were subjected to functional genomic studies. Electromobility shift assay (EMSA) showed that the rs73748206 “T” allele altered DNA-protein binding patterns, consistent with a significantly increased reporter gene activity. The effect of rs73748206 was also confirmed by mRNA expression studies using lymphoblastoid cell lines (LCL) derived from the same patients from whom DNA used for resequencing had been obtained. This comprehensive FKBP5 resequencing study provides insight into the role of inheritance in variation in response to gemcitabine therapy of pancreatic cancer. Citation Format: Katarzyna A. Ellsworth, Bruce W. Eckloff, Liang Li, Irene Moon, Brooke L. Fridley, Greg D. Jenkins, Erin Carlson, Abra Brisbin, Ryan Abo, William Bamlet, Gloria Petersen, Eric D. Wieben, Liewei Wang. Contribution of FKBP5 genetic variation to gemcitabine treatment and survival in pancreatic 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 2209. doi:10.1158/1538-7445.AM2013-2209

Abstract The pyrimidine analog 5-fluorouracil (5-FU) was developed in the 1950s and remains one of the most widely used chemotherapy drugs in use today. A significant portion of patients who receive 5-FU suffer severe adverse toxicities, and, in extreme cases, death. Dihydropyrimidine dehydrogenase (DPD, encoded by the DPYD gene) rapidly degrades 85% of administered 5-FU, and as such, limits the amount of drug available for conversion into active metabolites. Genetic variations in DPYD have been suggested to increase the risk of 5-FU toxicity. Large-scale sequencing efforts have identified nearly 100 missense and nonsense DPYD variations, however only a handful of alleles have been studied for their association with 5-FU toxicity. Most studies have focused on a single allele (DPYD*2A), which only accounts for a small fraction of DPD deficiencies. Further complicating the interpretation of DPYD alleles, many individuals carry multiple variations within the DPYD gene. The purpose of this study was to identify missense variants that affect the enzyme function of DPD using a mammalian isogenic system of expression. Expression vectors encoding missense DPYD variations were generated by site directed mutagenesis and the expressed protein assayed for enzyme activity. Cells expressing selected variants were also teasted for sensitivity to 5-FU using a novel implementation of electrical impedance based realtime cellular analysis. The results of functional studies are compared to in silico predictions used to classify variants as deleterious or benign. An appreciable fraction of DPD variants showed enzyme activity that was significantly lower than that of wildtype protein. Within the deficient group of variants, a gradient of activities was noted, prompting the grouping of variants as mildly, moderately, and severely impaired. In addition to loss-of-function variations, a few hyperactive DPYD alleles were identified. This novel class of DPYD variants may be protective against 5-FU toxicity at the cost of treatment efficacy. The further evaluation of hyperactive alleles in clinical studies will be necessary to determine their impact on patient outcomes. Overall these data will facilitate the development of predictive tests for 5-FU toxicity by allowing researchers to prioritize variations for association studies. Citation Format: Steven M. Offer, Natalie J. Wegner, Croix Fossum, Robert D. Diasio. Classification of DPYD variations using an isogenic system of expression. [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 2202. doi:10.1158/1538-7445.AM2013-2202

Abstract The plasma cell malignancy Multiple Myeloma (MM) is a rare but deadly form of cancer with 5 year survival rates of ∼30%. Epidemiological studies have suggested that MM exacts an especially heavy burden on African American (AA) patients. We examined a multi-ethnic cohort of MM tumors to assess potential differences in the frequency of molecular events in tumors derived from either African American (AA) or European American (EA) patients. The frequency of 14q32 translocation breakpoints at the IgH locus was compared among a series of data from 115 AA MM patients from three studies and EA MM from the Eastern Cooperative Oncology Group (ECOG). In addition, we analyzed matching genome-wide copy number and transcriptional data among 45 AA MM tumors and 196 EA MM tumors to determine differences in the frequency of molecular events in tumors from these populations. No differences were found for specific translocation subtypes; however, we detected a statistically significant difference in the overall frequency of IgH translocations between the two groups, which occurred more frequently in EA patients (52% vs. 40%; p = 0.032). When assessing genomic copy number events previously shown to be associated with poor outcome in MM, frequencies of alterations were not significant after correcting for multiple testing. Furthermore, there was not a significant difference in the association of high-risk disease using expression profiling. Our study represents the first comprehensive assessment of the frequency and distribution of molecular alterations in MM tumors from both AA and EA patients and could help shed light on possible biological features associated with population differences in incidence and outcome in MM. Citation Format: Angela S. Baker, Esteban Braggio, Susana Jacobus, Sungwon Jung, Dirk Larson, Terry Therneau, Angela Dispenzieri, Scott A. Van Wier, Gregory Ahmann, Joan Levy, Louise Perkins, Seungchan Kim, Kim Henderson, David Vesole, S. Vincent Rajkumar, Dianne F. Jelinek, John Carpten, Rafael Fonseca. Characterization of myeloma tumors from a multi-ethnic cohort using cytogenetics and genomic analysis. [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 2021. doi:10.1158/1538-7445.AM2013-2021

Abstract Next Generation Sequencing (NGS) technologies provide rapid genomic analyses of single nucleotide variants, RNA expression and DNA copy number. Application of these technologies to material isolated from formalin fixed paraffin embedded (FFPE) tissue and even degraded frozen material could provide powerful replication samples but remains challenging. We tested the nanoString platform to validate deep sequence analysis of gene expression and DNA copy number in degraded and FFPE material. Firstly, RNA from the Universal Human Reference RNA and a breast cancer cell line (MDA-MB-436) was artificially degraded to different degrees (RIN 1.2-6.8). We used the nanoString platform to simultaneously measure RNA expression across 226 genes in each degraded sample and the corresponding undegraded RNA. Secondly we isolated RNA and DNA from matched fresh frozen and FFPE tissues from nine breast cancer patients (3 HER2+/ER+/PR+, 2 HER2+/ER-/PR-, 2 HER2+/ER+/PR-, 2 HER2-/ER+/PR+) using the nanoString platform to compare expression and copy number across 226 and 86 genes respectively. Finally, we correlated expression and copy number data generated by nanoString with Illumina transcriptome and whole genome sequencing (WGS). NanoString log2 expression fold-change between all artificially degraded samples and their undegraded counterpart showed extremely high correlation (r2>0.91). NanoString DNA copy number between matched fresh-frozen and FFPE showed a high degree of correlation (r2=0.71). All gene amplifications with copy number ≥ 5 in DNA from fresh-frozen material (N=9) were successfully identified in DNA from FFPE material. We also observed good correlation of gene expression between whole transcriptome sequencing and the nanoString platform (r2 0.59 - 0.72) in FFPE and artificially degraded material and for DNA copy number between WGS and nanoString in DNA isolated from cancer cell lines (r2=0.96). The nanoString platform provides reliable data from highly degraded and FFPE material and correlates with sequence analysis of both expression and copy number from NGS platforms demonstrating potential for large-scale replication studies in FFPE material. Citation Format: Nadine Norton, Edith A. Perez, Yan W. Asmann, Jennifer M. Carr, Brian M. Necela, Jennifer M. Kachergus, Jin Jen, Bruce W. Eckloff, E Aubrey Thompson. Analysis of gene expression and copy number variation in breast tumors using both sequencing and hybridization-based platforms. [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 2005. doi:10.1158/1538-7445.AM2013-2005

Abstract 15 - 30 % of all breast cancer cases are classified as HER2/neu-positive, a very aggressive form of breast cancer that correlates with a poor prognosis, reduced survival time and increased incidence of metastasis. Kourtidis et al. (2010) have recently shown that the HER2-positive breast cancer cell line BT474 relies on a unique Warburg-like metabolism for survival and aggressive behavior. These cells are dependent on fatty acid synthesis, presumably as a means to regenerate NAD+ for aerobic glycolysis. The nuclear receptors NR1D1 and PPARγ enable these cells to upregulate de novo fatty acid synthesis while avoiding palmitate-induced lipotoxicity by storing neutral fats (TAGs). The cells show markedly increased levels of stored fats compared to other breast cancer cell lines and disruption of the synthetic process results in apoptosis of BT474 cells. Metabolomic analysis of NR1D1 overexpressing cells compared to normal cells reveals profound differences in their metabolite profiles. This metabolic phenotype is operating at its limits in HER2-positive cells, as addition of exogenous palmitate results in cell death, whereas HER2-normal cells are not affected by this treatment. Microarray analysis suggests that palmitate induces an ER-stress response in the HER2-positive cells, ultimately resulting in cell death. Preliminary results suggest that this response is CHOP-dependent. Combination treatments with palmitate and HER2-targeted therapeutic compounds show synergistic effects and markedly increase the cytotoxic effects of either compound alone. The uptake of exogenous fatty acids as well as the de novo fatty acid synthesis are dependent on the essential co-factor coenzyme A (CoA), which is generated from the precursor pantothenate (vitamin B5). Based on our genomic analysis we predicted that HER2-positive breast cancer cells are more sensitive to changes in intracellular CoA levels. Our results show that HER2-positive breast cancer cells are sensitive to shRNA-mediated knockdown of coenzyme A pathway enzymes whereas MCF-7 cells are not. We observe the same pattern of sensitivity using the competitive CoA pathway inhibitor homopantothenate. Interestingly, limiting the supply of CoA in HER2-positive cells reduces the toxicity of exogenous palmitate. Our data suggest that HER2-positive breast cancer cells are metabolically different from other breast cancer cell lines and that these differences offer new possibilities for therapeutic applications. Citation Format: Jan M. Baumann, Antonis Kourtidis, Douglas S. Conklin. The lipogenic phenotype of HER2/neu-positive breast cancer cells. [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 1894. doi:10.1158/1538-7445.AM2013-1894

Abstract Hydrogen sulfide (H2S), the third gasotransmitter, has recently attracted a lot of attention for its cytoprotective action and role in angiogenesis. Dysregulation of the major enzymes which produces H2S namely, cystathionine beta synthase (CBS), cystathionine gamma lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST) have also been implicated in a large number of pathological conditions that range from neurological disorders to cardiovascular diseases. Surprisingly, studies on the role of these enzymes in cancer is scarce, especially, that of CBS. Relapse with a drug resistant and aggressive phenotype is a common occurrence in ovarian cancer with an overall 5-year survival of approximately 30%. Here we report that, CBS, a key enzyme required for sulphur amino acid metabolism, plays a critical role in promoting ovarian cancer growth, metastasis and drug resistance. Overexpression of CBS was observed in a majority of ovarian cancer cell lines utilized in our study compared to normal ovarian surface epithelial cells. Silencing CBS in A2780 cells using siRNA or a CBS-specific inhibitor, aminooxyacetic acid reduced proliferation of ovarian cancer cells by dramatically reducing total glutathione levels, H2S levels and thereby enhancing oxidative stress. Downregulation of CBS reduced activation of NF-kB and upregulated p53 protein levels thereby inactivating the antioxidant network of the cells. A deeper insight into the intracellular localization of CBS by confocal fluorescence microscopy demonstrated a mitochondria specific distribution and silencing CBS caused mitochondrial superoxide accumulation that resulted in decreased mitochondrial respiration, reduced ATP production and increased ADP-to-ATP ratio. Furthermore, silencing CBS, augmented the activity of cisplatin in vitro in A2780 cells and in vivo in an orthotopic model of advanced ovarian cancer. Nanoliposomal delivery of CBS siRNA significantly reduced the number of tumor nodules in a chemoresistant orthotopic mouse model of ovarian cancer. To our knowledge, this is the first study demonstrating the importance of CBS in ovarian cancer progression and drug resistance and therefore is a viable target for future anti-cancer therapies. Citation Format: Sounik Saha, Sanjib Bhattacharyya, Karuna Giri, Ian R. Lanza, K. Sreekumar Nair, Nicholas B. Jennings, Rakesh Kumar, Anil K. Sood, Resham Bhattacharya, Priyabrata Mukherjee. Role of cystathionine beta-synthase in ovarian cancer growth, metastasis and drug sensitivity. [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 1882. doi:10.1158/1538-7445.AM2013-1882

Abstract Prostate carcinogenesis is a complicated process involving both genetic and epigenetic changes. Aberrant expression of LSD1 (Lysine-specific demethylase 1) has been reported in human prostate cancers. Although high expression of LSD1 is implicated in prostate tumorigenesis, the underlying molecular mechanisms are not fully understood. Here we reported that LSD1 promotes proliferation and migration of prostate cancer cells via up-regulation of SKP2 expression. Knockdown of LSD1 using small interfering RNA (siRNA) decreases SKP2 expression and inhibits prostate cancer cell proliferation and migration. Promoter luciferase reporter and chromatin immunoprecipitation (ChIP) experiments show that LSD1 regulates SKP2 expression by controlling the levels of H3K4 methylation in SKP2 promoter and this function of LSD1 is mediated through its demethylase activity. As demonstrated by immunohistochemistry, LSD1 and SKP2 proteins positively correlate with each other and their expression is associated with cell proliferation in human prostate cancer tissues. Our data not only identify SKP2 as a direct regulated target gene of LSD1 but also reveal that SKP2 plays an important role in contributing LSD1 mediated prostate cancer cell proliferation and migration, suggesting that the LSD1-SKP2 axis can serve as therapeutic targets for treatment of prostate cancers. Citation Format: Liya Ding, Haojie Huang. LSD1 regulation of SKP2 expression 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 1805. doi:10.1158/1538-7445.AM2013-1805

Abstract PURPOSE: Despite the development of prognostic algorithms based on clinico-pathologic features, the ability to identify aggressive forms of clear cell renal cell carcinoma (ccRCC) remains suboptimal. Topoisomerase IIA (TOP2a) is a biomarker of DNA replication and a target for antineoplastic agents. Herein, we evaluate the association of TOP2a expression in ccRCC tumors with pathologic features of aggressiveness and risk of cancer-specific death. METHODS: We identified 947 patients who underwent nephrectomy to treat clinically localized ccRCC between 1/16/1990 and 9/27/2006. TOP2a expression was assessed using IHC and scored as number of positive cells per mm2. We evaluated TOP2a expression using a continuous variable and tertile categories. For associations with pathologic features we employed Kruskal-Wallis tests and for associations with cancer-specific survival we generated Cox proportional hazard regression models. RESULTS: Higher TOP2a expression is associated with later stage, higher grade and higher Mayo SSIGN score (all p < 0.001). The risk of death from RCC increases with increasing TOP2a expression (p trend < 0.0001). Compared to patients in the lowest tertile, those patients with tumors in the highest tertile of TOP2a expression were at increased risk of RCC death (HR=2.31 95% CI 1.64-3.25; p<0.0001). Interestingly, among those patients with low risk disease (SSIGN score 0-3; ∼95% 10 year survival), those with high TOP2a were at increased risk of RCC death (HR=3.09 95% CI 1.29-7.40; p =0.01). CONCLUSION: Higher TOP2a expression is associated with more aggressive pathologic features and increased risk of cancer-specific death among patients undergoing surgery for localized ccRCC. If confirmed, these data support further inquiry for TOP2a as a prognostic and predictive biomarker for ccRCC patients. Citation Format: Richard W. Joseph, Dan Serie, Tracy Hilton, Mansi Parasramka, Jeanette Eckel-Passow, John Cheville, Bradley C. Leibovich, Alexander S. Parker. Association of topoisomerase II expression and cancer-specific death in patients with surgically-resected clear cell renal 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 15. doi:10.1158/1538-7445.AM2013-15