Dylan M. Glubb

A genome-wide association study of overall survival in pancreatic cancer patients treated with gemcitabine in CALGB 80303

Background and Aims: Cancer and Leukemia Group B 80303 was a randomized, phase III study in patients with advanced pancreatic cancer treated with gemcitabine plus either bevacizumab or placebo. We prospectively collected germline DNA and conducted a genome-wide association study (GWAS) using overall survival (OS) as the endpoint. Experimental Design: DNA from 351 patients was genotyped for more than 550,000 single-nucleotide polymorphisms (SNP). Associations between OS and SNPs were investigated using the log-linear 2-way multiplicative Cox proportional hazards model. The subset of 294 genetically European patients was used for the primary analysis. Results: A nonsynonymous SNP in interleukin (IL)17F (rs763780, H161R) and an intronic SNP in strong linkage disequilibrium (rs7771466) were associated with OS using genome-wide criteria (P ≤ 10−7). Median OS was significantly shorter (P = 2.61 × 10−8) for the rs763780 heterozygotes [3.1 months; 95% confidence interval (CI), 2.3–4.3] than for the patients without this variant (6.8 months; 95% CI, 5.8–7.3). After adjustment by stratification factors, the P value for the association was 9.51 × 10−7. Conclusions: The variant 161R form of IL-17F is a natural antagonist of the antiangiogenic effects of wild-type 161H IL-17F, and angiogenesis may play an important role in the metastatic spread of pancreatic cancer. In this preliminary study, we hypothesize that the angiogenetic potential of pancreatic cancers in patients with variant IL-17F is higher than that of tumors in patients with wild-type IL-17F, conferring worse prognosis. This exploratory GWAS may provide the foundation for testing the biology and clinical effects of novel genes and their heritable variants through mechanistic and confirmatory studies in pancreatic cancer. Clin Cancer Res; 18(2); 577–84. ©2011 AACR.

Novel Functional Germline Variants in the VEGF Receptor 2 Gene and Their Effect on Gene Expression and Microvessel Density in Lung Cancer

Purpose: VEGF receptor 2 (VEGFR-2) plays a crucial role in mediating angiogenic endothelial cell responses via the VEGF pathway, and angiogenesis inhibitors targeting VEGFR-2 are in clinical use. As angiogenesis is a host-driven process, functional heritable variation in KDR, the gene encoding VEGFR-2, may affect VEGFR-2 function and, ultimately, the extent of tumor angiogenesis. Experimental Design: We resequenced KDR using 24 DNAs each from healthy Caucasian, African American, and Asian groups. Nonsynonymous genetic variants were assessed for function by phosphorylation assays. Luciferase reporter gene assays were used to examine effects of variants on gene expression. KDR mRNA and protein expression and microvessel density (MVD) were measured in non–small cell lung cancer (NSCLC) tumor samples, and matching patient DNA samples were genotyped to test for associations with variants of interest. Results: KDR resequencing led to the discovery of 120 genetic variants, of which 25 had not been previously reported. Q472H had increased VEGFR-2 protein phosphorylation and associated with increased MVD in NSCLC tumor samples. −2854C and −2455A increased luciferase expression and associated with higher KDR mRNA levels in NSCLC samples. −271A reduced luciferase expression and associated with lower VEGFR-2 levels in NSCLC samples. −906C and 23408G associated with higher KDR mRNA levels in NSCLC samples. Conclusions: This study has defined KDR genetic variation in 3 populations and identified common variants that impact on tumoral KDR expression and vascularization. These findings may have important implications for understanding the molecular basis of genetic associations between KDR variation and clinical phenotypes related to VEGFR-2 function. Clin Cancer Res; 17(16); 5257–67. ©2011 AACR.

Mechanisms of genetic regulation in gene expression: examples from drug metabolizing enzymes and transporters

Interindividual variability in the response to drug therapy is due, in part, to genetic mechanisms which influence the expression of genes involved with drug metabolism and transport. Genetic elements and processes such as DNA methylation, histone deacetylation, transcription factors, DNA sequence variants, and microRNAs (miRNAs) can impact at either the transcriptional or translational levels to modulate gene expression. Identification of such genetic regulators has greatly advanced in the last decade. Genome‐wide analyses, using different types of approaches and methodologies, have uncovered many potential regulators of the expression of drug metabolizing enzymes and transporters. However, confirming the function of these putative regulators is necessary and requires further work in the laboratory, using techniques which are still evolving. It also still remains to be seen whether these findings have clinical implications for drug therapy but the realization of personalized medicine is a possible consequence of this research. WIREs Syst Biol Med 2011 3 299–313 DOI: 10.1002/wsbm.125

Liver expression quantitative trait loci: a foundation for pharmacogenomic research

Expression quantitative trait loci (eQTL) analysis can provide insights into the genetic regulation of gene expression at a genomic level and this information is proving extremely useful in many different areas of research. As a consequence of the role of the liver in drug metabolism and disposition, the study of eQTLs in primary human liver tissue could provide a foundation for pharmacogenomics. Thus far, four genome-wide eQTL studies have been performed using human livers. Many liver eQTLs have been found to be reproducible and a proportion of these may be specific to the liver. Already these data have been used to interpret and inform clinic genome-wide association studies, providing potential mechanistic evidence for clinical associations and identifying genes which may impact clinical phenotypes. However, the utility of liver eQTL data has not yet been fully explored or realized in pharmacogenomics. As further liver eQTL research is undertaken, the genetic regulation of gene expression will become much better characterized and this knowledge will create a rational basis for the prospective pharmacogenomic study of many drugs.

Architecture of pharmacogenomic associations: structures with functional foundations or castles made of sand?

In the construction of any building, a foundation beneath the surface is required to ensure that the structure has a stable footing and is serviceable. Just like a building, a pharmacogenomic association requires a foundation. In this case, a foundation can be constructed from the elucidation of the genetic mechanism behind the pharmacogenomic association, providing a supportive biological basis. If the function of the genetic variant believed to provide the association (which we will call ‘associated SNP’ for simplicity) is not known, it is difficult to make a biological interpretation and determine whether the causal variant has been identified [1]. Consequently, establishing a functional foundation is a crucial step in the validation of any pharmacogenomic association and even more so when the ability to conduct external replication is limited. This is particularly a problem of pharmacogenomic studies stemming from clinical trials of experimental drugs, as independent study cohorts will very likely not exist. In these instances, the establishment of a functional foundation is an essential part in the process of demonstrating clinical validity and utility.

Discovery and Functional Assessment of Gene Variants in the Vascular Endothelial Growth Factor Pathway

Angiogenesis is a host‐mediated mechanism in disease pathophysiology. The vascular endothelial growth factor (VEGF) pathway is a major determinant of angiogenesis, and a comprehensive annotation of the functional variation in this pathway is essential to understand the genetic basis of angiogenesis‐related diseases. We assessed the allelic heterogeneity of gene expression, population specificity of cis expression quantitative trait loci (eQTLs), and eQTL function in luciferase assays in CEU and Yoruba people of Ibadan, Nigeria (YRI) HapMap lymphoblastoid cell lines in 23 resequenced genes. Among 356 cis‐eQTLs, 155 and 174 were unique to CEU and YRI, respectively, and 27 were shared between CEU and YRI. Two cis‐eQTLs provided mechanistic evidence for two genome‐wide association study findings. Five eQTLs were tested for function in luciferase assays and the effect of two KRAS variants was concordant with the eQTL effect. Two eQTLs found in each of PRKCE, PIK3C2A, and MAP2K6 could predict 44%, 37%, and 45% of the variance in gene expression, respectively. This is the first analysis focusing on the pattern of functional genetic variation of the VEGF pathway genes in CEU and YRI populations and providing mechanistic evidence for genetic association studies of diseases for which angiogenesis plays a pathophysiologic role.

Functional FLT1 Genetic Variation is a Prognostic Factor for Recurrence in Stage I–III Non–Small-Cell Lung Cancer

Background: We propose that single-nucleotide polymorphisms (SNPs) in genes of the vascular endothelial growth factor pathway of angiogenesis will associate with survival in non–small-cell lung cancer (NSCLC) patients. Methods: Fifty-three SNPs in vascular endothelial growth factor-pathway genes were genotyped in 150 European stage I–III NSCLC patients and tested for associations with patient survival. Replication was performed in an independent cohort of 142 European stage I–III patients. Reporter gene assays were used to assess the effects of SNPs on transcriptional activity. Results: In the initial cohort, five SNPs associated (q < 0.05) with relapse-free survival (RFS). The minor alleles of intronic FLT1 SNPs, rs7996030 and rs9582036, associated with reduced RFS (hazard ratio [HR] = 1.67 [95% confidence interval, CI, 1.22–2.29] and HR = 1.51 [95% CI, 1.14–2.01], respectively) and reduced transcriptional activity. The minor alleles of intronic KRAS SNPs, rs12813551 and rs10505980, associated with increased RFS (HR = 0.64 [0.46–0.87] and HR = 0.64 [0.47–0.87], respectively), and the minor allelic variant of rs12813551 also reduced transcriptional activity. Lastly, the minor allele of the intronic KRAS SNP rs10842513 associated with reduced RFS (HR = 1.65 [95% CI, 1.16–2.37]). Analysis of the functional variants suggests they are located in transcriptional enhancer elements. The negative effect of rs9582036 on RFS was confirmed in the replication cohort (HR = 1.69 [0.99–2.89], p = 0.028), and the association was significant in pooled analysis of both cohorts (HR = 1.67 [1.21–2.30], p = 0.0001). Conclusions: The functional FLT1 variant rs9582036 is a prognostic determinant of recurrence in stage I–III NSCLC. Its predictive value should be tested in the adjuvant setting of stage I–III NSCLC.

The Vitamin D receptor gene as a determinant of survival in pancreatic cancer patients: Genomic analysis and experimental validation

Purpose Advanced pancreatic cancer is a highly refractory disease almost always associated with survival of little more than a year. New interventions based on novel targets are needed. We aim to identify new genetic determinants of overall survival (OS) in patients after treatment with gemcitabine using genome-wide screens of germline DNA. We aim also to support these findings with in vitro functional analysis. Patients and methods Genome-wide screens of germline DNA in two independent cohorts of pancreatic cancer patients (from the Cancer and Leukemia Group B (CALGB) 80303 and the Mayo Clinic) were used to select new genes associated with OS. The vitamin D receptor gene (VDR) was selected, and the interactions of genetic variation in VDR with circulating vitamin D levels and gemcitabine treatment were evaluated. Functional effects of common VDR variants were also evaluated in experimental assays in human cell lines. Results The rs2853564 variant in VDR was associated with OS in patients from both the Mayo Clinic (HR 0.81, 95% CI 0.70–0.94, p = 0.0059) and CALGB 80303 (HR 0.74, 0.63–0.87, p = 0.0002). rs2853564 interacted with high pre-treatment levels of 25-hydroxyvitamin D (25(OH)D, a measure of endogenous vitamin D) (p = 0.0079 for interaction) and with gemcitabine treatment (p = 0.024 for interaction) to confer increased OS. rs2853564 increased transcriptional activity in luciferase assays and reduced the binding of the IRF4 transcription factor. Conclusion Our findings propose VDR as a novel determinant of survival in advanced pancreatic cancer patients. Common functional variation in this gene might interact with endogenous vitamin D and gemcitabine treatment to determine improved patient survival. These results support evidence for a modulatory role of the vitamin D pathway for the survival of advanced pancreatic cancer patients.

Abstract 5136: Discovery of novel functional germline variations in the vascular endothelial growth factor receptor 2 gene (KDR) and their effect on gene expression and microvessel density in lung cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Background: VEGFR-2 plays a crucial role in mediating angiogenic endothelial cell responses via the VEGF pathway and VEGFR-2 inhibitors are in clinical use. Angiogenesis is a host-driven process and heritable variation in KDR, the gene encoding VEGFR-2, may impact on angiogenesis. We have previously characterized KDR germline genetic variation by resequencing (Ye et al., AACR, 2007;737). However, very limited data are available on the molecular function of KDR germline variants and their resulting effect on tumoral expression and vascularization. The purpose of this study was to identify functional variants affecting KDR expression and microvessel density (MVD) in lung cancer. Methods: The molecular function of the non-synonymous variants R106W, V297I, Q472H and C482R was assessed by testing their effect on VEGFR-2 phosphorylation (measured via immunoblotting) after VEGFA165 exposure in HEK293 cells. Four 5’ upstream region SNPs were examined by luciferase reporter assay in endothelial SVEC4-10 cells. To validate these results and examine the effects of other KDR SNPs, KDR mRNA and VEGFR-2 protein expression were measured by quantitative PCR and immunohistochemistry in non-small cell lung cancer (NSCLC) samples from Caucasian patients and MVD was assessed by immunohistochemistry (anti-CD31). Patients were genotyped for KDR SNPs and their association with mRNA, protein expression, or MVD was evaluated by linear regression. Results: Among the non-synonymous SNPs, the only significant observation was that Q472H had a 46% increase in VEGFR-2 phosphorylation relative to the wild-type variant after VEGFA165 stimulation (p=0.035); in agreement with this finding, Q472H was associated with 22% greater MVD in 168 NSCLC patients (p=0.05). Of the putatively regulatory SNPs, -2008G and -1942G had no effect on luciferase expression. -2854C and -2455A showed 10-20% higher luciferase expression than the wild-type variant (p<0.05) and they were associated with 23% greater tumor KDR mRNA expression in 66 NSCLC patients (p=0.014), but had no effect on tumor VEFGR-2 expression; -271A reduced luciferase expression by approximately 50% (Ye et al., AACR 2007;737) and correlated with 10% lower tumor VEGFR-2 expression in 162 NSCLC patients (p=0.01); -906C and 23408G were associated with 20-25% higher tumor KDR mRNA levels (p=0.009 and 0.025, respectively), although these variants had no effect on tumor VEGFR-2 expression. Conclusions: This study has identified novel functional variants which correlate with in vitro gene expression or VEGFR-2 phosphorylation and KDR expression or MVD in NSCLC. This information may be critical for understanding the role of heritable KDR variations as contributing factors to prognosis and lung cancer outcome. Acknowledgment: This research was supported by the American Cancer Society. 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 5136. doi:10.1158/1538-7445.AM2011-5136

Abstract 5148: Non-small cell lung cancer expression ofVEGF pathway genes and association with heritable genetic variants

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Background: Heritable functional genetic variation in the VEGF pathway may impact on tumor angiogenesis and, hence, clinical cancer outcomes. However, there is very limited data currently available on which common germline variants affect gene expression in the tumor vasculature. Thus, the purpose of this study is to fill this critical gap in the knowledge. To achieve this aim, we used tumor samples to characterize the mRNA expression of several key genes in the VEGF pathway and then examined genetic associations with candidate SNPs. We have used several different approaches to generate a list of candidate SNPs for analysis, including: the use of bioinformatic tools, our previous study of gene resequencing of VEGF-pathway genes and genetic associations with mRNA levels in lymphoblastoid cell lines (LCLs) (Pare-Brunet, AACR 2008;#4332), and the identification of functional variants from the literature. Methods: The mRNA expression of HIF1A, KRAS (isoforms A and B), PGF, VEGFA (total and plus the 165 isoform) and VEGFB was measured by quantitative PCR in 91 European NSCLC specimens and mRNA levels were normalized using the expression of 18S. Common putatively regulatory or functional SNPs in these genes were identified using our previous study, the FastSNP bioinformatic web tool (http://fastsnp.ibms.sinica.edu.tw), and literature searches. Candidate SNPs were genotyped in the NSCLC cohort using TaqMan assays and genotype-expression associations were analyzed by linear regression, correcting for clinical characteristics of patients. Results : We quantified NSCLC tumoral mRNA expression and found PGF mRNA levels were significantly greater (p<0.001) than those of any other gene and were followed in decreasing order of expression by VEGFB, KRAS (A and B isoforms), VEGFA (total and 165 isoform) and HIF1A. We genotyped 14 SNPs using matching DNA samples and three SNPs significantly associated with mRNA levels in NSCLC tumors: rs10842513 (minor allele frequency (MAF)=0.10), rs3025006 (MAF=0.32) and rs1573060 (MAF=0.38). rs10842513 correlated with higher KRAS (isoform B) mRNA levels after controlling for tumor histology (p=0.007), in agreement with the association between this variant and KRAS levels in LCLs; rs3025006 associated with lower total VEGFA expression (p=0.020); and rs1573060 correlated with both lower total VEGFA levels (p=0.021), as well as with lower expression of the 165 isoform (p=0.024). Conclusion: We have successfully found common genetic variants which associate with mRNA expression from tumor samples. This study provides crucial knowledge as functional genetic variants may affect clinical cancer outcomes. Indeed, one of SNPs we identified, rs10842513, is known to associate with poorer survival in ovarian cancer (Quaye et al., Clin Cancer Res 2008;14: 5833). Such clinical associations will be examined in future studies. 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 5148. doi:10.1158/1538-7445.AM2011-5148