Citations

Abstract Phospholipase C epsilon (PLCε or PLC∈ 1) is a phosphoinositide-specific enzyme phospholipase C that converts phosphatidylinositol 4,5-biphosphate to two intracellular second messengers, diacylglycerol and inositol 1,4,5-trisphosphate, which regulate protein kinase C activity and calcium mobilization, respectively. These responses are involved in the regulation of cell growth, differentiation and oncogenesis. However, the involvement of PLCε in human carcinogenesis, particularly in esophageal cancer, was not been recognized until a susceptible SNP (rs2274223: A5780G:His1927Arg) was recently identified in a large genome-wide association study (GWAS). This SNP, located within exon 26 and C2 domain of the PLCε gene at chromosome 10q23, leads to a nonsynonymous alteration from CAC (A allele, encoding histidine) to CGC (G allele, encoding arginine), which was associated with an increased risk of both squamous cell carcinoma and esophagus-cardia gastric adenocarcinoma. In the current report, we demonstrate that the presence of the G allele (AG or GG) at this locus leads to increased PLCε mRNA and protein expression in human esophageal cancer tissues and in human esophageal cancer cell lines. The present of G allele also leads o a higher enzyme activity in esophageal cancer cells in vitro. Quantitative analysis of the C2 domain sequences revealed that A:G allelic imbalance is strongly associated with esophageal malignancy. Moreover, the analysis of 10,614 non-cancer subjects from high- and low-risk areas of China demonstrated that the presence of the G allele is strongly associated with moderate to severe esophagitis in the subjects from the high-risk areas of China: 77% of the severe esophagitis individuals in high-risk areas had AG/GG genotypes vs. only 37% of these subjects in low-risk areas (OR 6.03 with 95% CI 1.59-22.9 vs. OR 0.74 with 95% CI 0.33-1.64; p=0.008). These data suggest that the interaction of potential environmental factors with PLCε, particularly in individuals with AG or GG allele, not only exists in high-risk areas for esophageal cancer development in China, but also correlates with the severity of esophagitis. In conclusion, PLCε is likely to play a pivotal role in esophageal carcinogenesis: the presence of the 5780G allele may not only predict a high risk of future esophageal cancer development, but may also participate in esophageal cancer growth and progression by upregulating levels of PLCε mRNA, protein, and enzyme activity, ultimately leading to augmentation of the inflammatory process in esophageal epithelium. Thus, the 5780G allele in PLCε may constitute a promising biomarker for esophageal squamous carcinoma risk stratification, early detection, and progression prediction. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4704. doi:10.1158/1538-7445.AM2011-4704

Abstract DNA double-strand breaks (DSBs) are highly cytotoxic lesions caused by genotoxin such as ionizing radiation (IR). Following DSB, cells activate the DNA-damage response pathway by activating protein kinases such as ATM, ATR and DNA-PK, which then trigger histone H2AX phosphorylation and the accumulation of proteins such as Mediator of DNA damage checkpoint protein 1 (MDC1), 53BP1, and BRCA1 into IR-induced foci. This process initiates the repair of DSB. Previously we have identified a novel biomarker for radiation response, Bora, by using a genome-wide association approach using data generated with 300 human lymphoblastoid cell liens. Bora is known as a cofactor of Aurora-A, which is involved in mitosis regulation. During G2, Aurora-A, in complex with Bora, phosphorylates Plk1 at Thr-210. Activated Plk1 phosphorylates Cdc25C and Wee1, which induces activation of cyclin B-cdk1 complexes and promotes mitotic entry. In this study, we want to determine the underlying mechanisms by which Bora regulates response to IR. We found that knockdown of Bora in human cancer cell lines including HupT3, MIAPaCa-2 and Hela (siBora) exhibit increased clonogenic survival rate in response to IR, an observation that is opposite from the effect of Plk1 knockdown, suggesting a different mechanism involved. Knockdown of Bora also desensitize cancer cells to chemotherapeutic agents including gemcitabine and oxiplatin. This enhanced resistance to genotoxin is primarily due to increased DNA DSB repair. More than 90% of DSBs were repaired in siBora cells compared with 60% in control cells 8 hours after cell exposure to 2 Gy IR. Upon IR, more cells with Bora knockdown retained in G2-M phase compared with control. Furthermore, Bora directly interacts with BRCT domain of MDC1 via its C terminus, and over expression of Bora can abolish MDC1 foci formation induced by IR. All of these observations suggested a role of Bora in DNA damage response pathway and in turn, in chemo-radio resistance. Indeed, overexpression of the full length or C-terminus of Bora sensitized cancer cells to IR treatment, while the N-terminal Bora did not have significant effect on IR response. In summary, we have identified a novel function of Bora involved in DNA damage and repair pathway, which indicates a broad role of Bora in both tumorgenesis and chemo-radio resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2660. doi:10.1158/1538-7445.AM2011-2660

Abstract There are a number of human genes which span very large chromosomal regions. Forty genes span greater than 1 megabase and 200 are 500 Kb or larger. We and others have demonstrated that many of the largest genes are derived from within regions of profound genomic instability known as common fragile sites (CFSs). We have been studying oropharyngeal cancers which are derived from the base of tongue, tonsil and larynx. While the traditional etiologic factors underlying the development of this aggressive cancer is a history of smoking and drinking there are an increasing number of younger patients developing this cancer without these risk factors. The most likely cause of these cancers is the presence of human papillomavirus which we detect in over 90% of oropharyngeal cancers derived from the Mayo Clinic. In an attempt to better understand the molecular alterations that underlie oropharyngeal cancers arising from different risk factors we utilized the power of Next Generation sequencing on the Illumina GA IIx to compare the transcriptomes of 12 oropharyngeal cancers to patient matched normal tissue. We first observe a division of patients into two groups of oropharyngeal cancers: those caused by mutations and other alterations induced by a history of smoking and drinking and those associated with HPV infection caused by viral-induced genomic instability. We then examined the expression of the 200 largest human genes in the oropharyngeal tumors and find that each tumor has inactivated the expression of at least 20 different very large genes. There is a distinct pattern of inactivation of these genes in different oropharyngeal cancers depending upon the underlying etiology. Some of the genes most frequently inactivated include Dlg2 (the human homolog of the large discs tumor suppressor gene from drosophila and a mutation target in a number of other cancers); ERBB4 (Her4 and a member of the EGFR family), BAI3 (a brain specific angiogenesis inhibitor that is also mutated in multiple cancers), and ANK2 (mutated in long QT patients). Each of these genes may be functioning as tumor suppressors as has already been demonstrated for the two large CFS genes, FHIT and WWOX. The inactivation of so many of these genes in each oropharyngeal tumor may have a profound effect upon the phenotype of the resulting tumor. When we examine the expression of the large genes most frequently inactivated in each of the two groups of oropharyngeal cancers in panels of breast or lung cancer samples we find that we do not detect significant loss of expression of these genes. We believe that genomic instability promotes alterations in the unstable CFS regions but that there is selective inactivation of different large CFS genes in different cancers. The signature of which specific genes are inactivated may have phenotypic and clinical significance which we are just beginning to investigate. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3885. doi:10.1158/1538-7445.AM2011-3885

Abstract There is considerable clinical interest in the role that human papillomavirus (HPV) plays in the development of oropharyngeal cancers. A variety of assays are currently available to stratify patients based on HPV status prior to treatment. The most frequently utilized are PCR and In Situ hybridization (ISH) to detect the presence of HPV DNA sequences. The increasing clinical interest in determining HPV patient status relates to compelling evidence that a subset of patients with HPV positive tumors have better clinical outcomes. Evidence also suggests that patients with HPV positive tumors should be further divided into groupings based on levels of viral activity and that this may represent additional clinical information necessary to develop appropriate treatment plans. The most frequently applied assay to identify HPV activity in a patient sample is to detect the over-expression of the surrogate protein marker p16 by Immunohistochemisty (IHC), which has been shown to increase expression in response to HPV active infections in oropharyngeal cancer. An alternative technique to quantitatively detect the presence and level of transcriptional activity of HPV in tissue samples is to measure the expression of oncogenes E6 and E7. We have analyzed the fresh frozen tissue samples corresponding to matched tumor, normal and metastatic patient tissues using a quantitative PCR (qPCR) approach to determine the expression of HPV oncogenes and p16 gene expression. We observed that there are three groups of oropharyngeal tumors when grouped by HPV16 E6 and E7 gene expression. The first are the most rare and are tumors that contain no HPV sequences or expression of the E6 and E7 ocogenes. The second are tumors that contain HPV DNA sequences but very low expression of E6 or E7 and the third are tumors that contain HPV DNA sequence and robust expression of E6 and E7. Additionally, we found variable correlation between HPV16 E6 and E7 expression and p16 gene expression, suggesting that factors other than HPV oncogene expression may influence the expression of p16. Our results would indicate that the best assays for quantifying the activity of an HPV infection in oropharyngeal cancers would be those optimized to accurately measure either E6 and E7 transcripts or their protein products directly out of paraffin-embedded tissues. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3859. doi:10.1158/1538-7445.AM2011-3859

Abstract INTRODUCTION: Mammographic breast density is a strong risk factor for breast cancer (BC). We established the Mayo Mammography Health Study (MMHS) cohort study at the Mayo Clinic in Rochester, Minnesota (MN) to examine the association of breast density with BC. We evaluated the influence of the image acquisition technique on the density and BC association. METHODS: From October 2003 to September 2006, all women scheduled for screening mammography at the Mayo Clinic were invited to participate. Eligible women were residents of MN, Iowa (IA) or Wisconsin (WI); age 35+; and had no personal history of BC. A risk factor questionnaire, consent form and permission to link to tumor registries were obtained. Incident BC was identified through 2009 by linkage to the Mayo and state cancer registries. A case-cohort of all incident BCs and 2300 randomly selected women (the subcohort) were used to examine the association of breast density and BC using digitized film mammograms at the time of enrollment while controlling for the influence of the acquisition parameters (peak kilovoltage, milliampere-second, and compressed breast thickness). Two density measures were considered: a quantitative percent density (PD) measure estimated using the computer-assisted thresholding program, Cumulus (University of Toronto), and the 4-category clinical BI-RADS measure. Proportional hazards regression was used to calculate hazards ratios (HR) and 95% confidence intervals (CI) for BC associated with quartiles of PD (0-6.2%,6.3-14.9%, 15.0-25.7% and 25.8%+) and BI-RADS categories 1-4 (almost entirely fatty to extremely dense). All models included age, postmenopausal hormone use (PMH), BMI, and menopausal status. The influence of acquisition parameters was evaluated by examining models with and without their inclusion. RESULTS: A total of 20,982 (50%) women participated in MMHS; 1058 (5%) with a prior history of BC were excluded, for a total cohort of 19,924. Compared to nonresponders, responders were younger (57.5 vs. 58.4 yrs), more likely to have ever used PMH (45% vs. 33%), and more likely to have a BC family history (19% vs 16%). The case-cohort consisted of 317 incident cases and 2300 women in the subcohort. Women were excluded from the current analysis if PD could not be estimated or if acquisition parameters were not available, leaving 249 cases and 1937 in the subcohort. As expected, PD was associated with BC [HR (95% CI): 1.0 (REF), 2.1 (1.4-3.1), 3.0 (2.0-4.5), and 4.6 (3.0-7.0) for quartiles; p-trend<0.001]. Controlling for acquisition parameters attenuated the association [HR (95% CI): 1.0 (REF), 2.3 (1.5-3.4), 2.4 (1.6-3.7), and 3.0 (1.8-5.0) for quartiles; p-trend<0.001]. Results for BI-RADS density were similar to those for PD. CONCLUSION: This study confirms that breast density is a significant risk factor for BC and demonstrates that the acquisition technique confounds the density and BC risk association. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3716. doi:10.1158/1538-7445.AM2011-3716

Abstract Background: Candidate gene and candidate pathway-based pharmacogenomic studies have been used to investigate the inherited variability of prognosis of lung cancer treated with platinum-based chemoptherapy. In order to make it possible to conduct an ‘unbiased’ query across the entire genome, a genome-wide association study (GWAS) in 283 lymphoblastoid cell lines (LCLs) was carried out to help identify candidate SNPs associated with variation in cisplatin cytotoxicity. Furthermore, we set out to test the hypothesis that genetic variation resulting in differences in cisplatin cytotoxicity might influence the overall survival of patients with lung cancer treated with platinum-based antineoplastic agents. Material and Methods: We applied ethnically diverse Coriell “Human Variation Panel” LCLs for GWAS. Then, we picked 168 top significant SNPs selected from GWAS in 283 LCLs and genotyped these SNPs in 1183 lung cancer patients (222 SCLC and 961 NSCLC) who received platinum-based chemotherapy. Genotyping was performed using the Illumina Golden Gate platform. The association of SNPs with lung cancer overall survival was analyzed by Cox regression model after adjusting for disease stage. Functional validation of candidate genes was performed by siRNA screening in NSCLC cell lines CRL5872, H1299 and SCLC cell line CRL5823. Results: A total of 157 SNPs were successfully genotyped for 1183 lung cancer patient samples. The Cox regression analysis indicated that 9 and 10 different SNPs were associated with overall survival for NSCLC and SCLC patients, respectively (p value <0.05). Fifteen genes including 7 genes located in the region of 200kb up or downstream of 4 SNPs (rs11169748, rs2440915, rs5952066 and rs7620841) and 8 genes for which the expression was correlated with three SNPs (rs11169748, rs2440915 and rs5952066) were selected for siRNA screening. Knockdown of DAPK3 and METTL6, for which the expression levels were correlated with the SNPs rs11169748 and rs2440915, significantly decreased cisplatin sensitivity in lung cancer cell lines CRL5872, H1299 and CRL5823. Conclusions: This series of clinical and complementary laboratory-based functional studies identified several candidate genes and SNPs that might help predict treatment outcomes of platinum-based therapy in lung cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1285. doi:10.1158/1538-7445.AM2011-1285

Abstract Background: T-DM1, an antibody-drug conjugate composed of trastuzumab, a stable linker, and the cytotoxic agent DM1, is in development for the treatment of HER2−positive cancers. Single-agent T-DM1 3.6 mg/kg every 3 weeks (q3w) has demonstrated clinical activity in 2 phase II studies (TDM4258g and TDM4374g) in patients with pretreated HER2−positive MBC. The efficacy and safety of T-DM1 vs trastuzumab + docetaxel was investigated in patients with no prior MBC treatment in the randomized phase II study TDM4450g/BO21976. Here we report the PK of T-DM1 from that study and compare these data with those from studies that enrolled pretreated patients. Methods: In all 3 studies, PK parameters, including maximum concentration (Cmax), area under the concentration-time curve (AUC), terminal half-life (t½), steady-state volume of distribution (Vss), and clearance (CL) were estimated by noncompartmental analysis (NCA) for serum T-DM1, serum total trastuzumab (conjugated and unconjugated), and plasma DM1. The effects of baseline trastuzumab and HER2 extracellular domain (ECD) concentration on T-DM1 exposure were explored and the relationship between T-DM1 exposure and clinical response (objective response rate [ORR] and progression-free survival [PFS]) was modeled. Results: T-DM1 PK from evaluable patients enrolled in 3 studies are shown in Table 1. No significant correlations were observed between efficacy (as measured by ORR) and T-DM1 exposure (AUC, Cmax) after administration of T-DM1 to pretreated patients; efficacy-exposure analyses (ORR and PFS) for previously untreated patients will be presented. Patients with measurable concentrations of trastuzumab at baseline had a greater AUC during cycle 1; however, this did not impact ORR. Baseline circulating HER2 ECD concentrations also had no effect on ORR for pretreated patients. The impact of baseline trastuzumab and HER2 ECD concentrations on ORR and PFS in previously untreated patients will be presented. Conclusions: Single-agent T-DM1 has similar PK in patients who have received prior therapy for MBC and in those who have not. The PK of T-DM1 was not affected by prior trastuzumab treatment or by circulating HER2 ECD, and no significant correlations were observed between efficacy (ORR) and T-DM1 exposure (AUC, Cmax) or HER2 ECD for pretreated patients. The relationships between efficacy and T-DM1 exposure and HER2 ECD concentrations will be presented for patients without prior MBC treatment. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-12-13.

Abstract Variation in drug response results from a combination of factors that include differences in gender, ethnicity, and environment, as well as genetic variation that may result in differences in mRNA and protein expression. This article presents two integrative analytic approaches that make use of both genome-wide SNP and mRNA expression data available on the same set of subjects: a step-wise integrative approach and a comprehensive analysis using sparse canonical correlation analysis (SCCA). In addition to applying standard SCCA, we present a novel modification of SCCA which allows different weighting for the various pair-wise relationships in the SCCA. These integrative approaches are illustrated with both simulated data and data from a pharmacogenomic study of the drug gemcitabine. Results from these analyses found little overlap in terms of genes detected, possibly detecting different biological mechanisms. In addition, we found the proposed weighted SCCA to outperform its unweighted counterpart in detecting associations between the genomic features and phenotype. Further research is needed to develop and assess new integrative methods for pharmacogenomic studies, as these types of analyses may uncover novel insights into the relationship between genomic variation and drug response.

Abstract We have recently showed that the immunophilin FKBP5 (also known as FKBP5) as a scaffolding protein that can enhance PHLPP-AKT interaction and facilitate PHLPP-mediated dephosphorylation of AKT-Ser473, negatively regulating AKT activation in vitro. Therefore, FKBP5 might function as a tumor suppressor and levels of FKBP5 would affect cell response to chemotherapy. In the current study, we took a step forward by using pancreatic caner xenograft mice model to show that FKBP5−/− promotes tumor growth and renders resistance to gemcitabine and addition of AKT inhibitors have more significant sensitization effect on FKBP5−/− mice to gemcitabine treatment. The combination of triciribine and gemcitabine resulted in cooperative inhibition of pancreatic cancer cell proliferation and the growth of xenografts in vivo without increasing toxicity, as well as cooperative inhibition of AKT signaling pathways at concentrations that were ineffective as individual agents. These results provide evidence for future clinical studies designed to tailor the therapy based on the biomarker. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C15.

Abstract Introduction: Androstenedione is a precursor for estrogen biosynthesis catalyzed by aromatase. Plasma androstenedione concentrations in postmenopausal women with early stage breast cancer display large interindividual variation (Ingle et al., Cancer Res. 70:3278–86, 2010). We performed a GWAS to identify single nucleotide polymorphisms (SNPs) associated with plasma levels of androstenedione in 776 postmenopausal women with resected early stage breast cancer. Methods: Plasma androstenedione concentrations were measured by GC-MS/MS. GWAS was performed using Illumina Human610-Quad BeadChips. The GWAS results were controlled for population stratification (PS) by Eigenstrat analyses. Plasma androstenedione concentrations were associated with genotype by using a quasi-likelihood model with adjustment for PS, BMI, age and other clinical variables associated with androstenedione concentrations (p<0.01). Imputation using HapMap2 data was performed within 200 kb on either side of regions containing SNPs with p<E-05. Results: 563,945 SNPs were analyzed, followed by imputation. The genotyped SNP with the lowest p value, rs4736317 (p=4.61E-06), was on chromosome 8 near the CYP11B1 and CYP11B2 genes. This SNP had a multiplicative effect of 1.128 (95% Cl: 1.07, 1.19) and a MAF of 0.40. After imputation, 20 SNPs with apparent p<E-06 were observed in this same region, with the lowest p value for rs9773022 (p=8.1E-08). We then performed functional genomic studies, and observed that two of the SNPs with low p values (rs4736312, p=1.49E-05 and rs4736350, p=6.34E-06) created apparent Oct-1 transcription factor binding sites (TFBS). Differential nuclear protein binding of Oct-1 to probes for these SNPs was verified with EMS and supershift assays using nuclear extract from A549 and NCI-H295R cells. Treatment of these two cell lines with 10 nM androstenedione led to the disruption of Oct-1 binding with the A (wild type) allele on rs4736312. rs4736312 is located in the 3'-UTR of CYP11B1 15 bp distant from response elements for the progesterone receptor and the glucocorticoid receptor. We also demonstrated that CYP11B1 and CYP11B2 were induced by estradiol in U2OS cell lines that express ER receptor and by androstenedione in H295R cells that express the androgen receptor. Transfection of a reporter gene construct with the rs4736312 SNP sequences into COS-1 and H295R cells suppressed reporter gene activity for the variant SNP sequence as compared to wild type, an observation that may correlate with the higher plasma levels of androstenedione in women (549× 328 vs 410×208 mean×SD, M/ml) with the variant allele. Conclusion: We have identified a series of SNPs in a region of chromosome 8 in and around CYP11B1 and CYP11B2 that are associated with elevated plasma androstenedione concentrations in postmenopausal women with resected early stage breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C114.