- Email: [email protected]
1171 DietCompLyf Study – a Multi-centre UK Study on Breast Cancer – What Are the Dietary and Lifestyle Changes Following Diagnosis?
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european journal of cancer 48, suppl. 5 (2012) S25–S288
linkage analysis of the SWR/J × (SWR/J × Car-R) backcross population using high-density marker coverage provided by single nucleotide polymorphism (SNP)-array. Gene expression analysis of normal lung tissues from Car-R and SWR/J was performed; then, we tested the effects of candidate genes (Igfbp2 and Igfbp5) overexpression on clonogenicity of lung cancer lines. Finally, functional analysis of the Igfbp5 promoter alleles from SWR/J and BALB/cJ by luciferase reporter assay was performed. Results and Discussion: We identified the ‘lung and skin tumorigenesis modifier 1’ (Lsktm1) locus on chromosome 1 linked to both skin (LOD score = 3.93) and lung (LOD score = 8.74) tumorigenesis. The Lsktm1 locus was confirmed in a (SWR/J × BALB/cJ)F2 intercross population, indicating that BALB/cJ carries the same cancer resistance alleles of Car-R mouse. Two genes, Igfbp5 and Igfbp2, residing in this locus and belonging to the insulin-like growth factor binding protein family, were expressed at significantly higher levels in normal lung tissue from cancer-resistant Car-R mice than in cancer-susceptible SWR/J mice, and overexpression of the recombinant genes in two lung cancer cell lines significantly inhibited clonogenicity (P < 0.0001). Functional analysis of the Igfbp5 promoter alleles from SWR/J and BALB/cJ by luciferase reporter assay showed differential expression between the strains (P = 0.0001), suggesting a possible involvement of Igfbp5 promoter variants in modulating gene expression. Conclusion: Collectively, our results demonstrate that skin and lung tumors are influenced by a single polymorphic genetic locus and identify Igfbp5 and Igfbp2 as candidate modifier genes of lung tumorigenesis. 1171 DietCompLyf Study − a Multi-centre UK Study on Breast Cancer − What Are the Dietary and Lifestyle Changes Following Diagnosis? K.A. Perkins1 , R. Swann1 , J. Woodside2 , C. Robertson1 , S. Dutton3 , A. Mulligan4 , L. Velentzis5 , M. Keshtgar6 , A. Leathem7 , M. Dwek1 . 1 University of Westminster, Department of Molecular and Applied Biosciences, London, United Kingdom, 2 Queens University, Belfast, United Kingdom, 3 University of Oxford, Centre for Statistics in Medicine, Oxford, United Kingdom, 4 University of Cambridge, Institute of Public Health, Cambridge, United Kingdom, 5 Cancer Epidemiology Research Unit, Cancer Council New South Wales, Sydney, Australia, 6 Royal Free Hospital, Department of Surgery, London, United Kingdom Background: Evidence summarising the influences of dietary and lifestyle factors in prevention of breast cancer is now established, altering the focus of research toward learning the independent and combined effects of features including alcohol, polyunsaturated fat intakes and exercise regimes on breast cancer outcomes. Method: The DietCompLyf study recruited 3,390 female breast cancer patients from 56 NHS centres across the UK. Blood and urine samples were collected alongside information on lifestyle, diet, psychological well-being and quality of life annually for 5 years. Describing the DietCompLyf cohort using tumour biology and treatment profiles, participants’ geographical location, age and ethnicity, the demographics of this cohort are compared with UK breast cancer rates. Additional features specific to the cohort including the socioeconomic status, general health and alcohol consumption are considered alongside changes made to the diet and lifestyle since diagnosis. Multivariate analysis (multiple linear regression) on the changes in BMI and its causes was carried out. Results and Discussion: Recruitment was closed in September 2010 and the collection and validation of baseline data completed. 231 records were excluded for data violation. Provisional analyses of socio-demographic data on the final dataset (3159 women, ages 26−75 years) shows that 88% (2786) were recruited from England, 6% from Wales. This compares well with UK cancer registry data for breast cancer (83% and 5.4% respectively). Populations in Scotland (4.2%) and Northern Ireland (1.4%) were slightly under-represented. In this cohort, 68% live in urban environments, 90% are white, 77% mothers, 70% current alcohol drinkers, 9% smokers and 65% partake in exercise once a week or more at recruitment. The median BMI of this UK cohort is 25.5 kg/m2. Following diagnosis, BMI increased on average by 0.65 kg/m2 . The results of a multivariate analysis on post diagnosis changes in BMI alongside changes in diet, smoking, drinking and exercise habits − including the observations that only 6% of women who drink alcohol at diagnosis and 8% of women who were smoking at diagnosis subsequently give up, will be presented. Conclusion: DietCompLyf is a large UK cohort of breast cancer survivors, the diversity of which is presented here. This cohort provides the opportunity to look at the modifiable changes made by women following a diagnosis of breast cancer. Recurrence and mortality data on members of the cohort being collected will be analyzed with respect to modifiable lifestyle habits once the data are mature. The results will inform advice to patients.
Sunday 8 − Tuesday 10 July 2012
1172 Colorectal Cancer eQTLs as Susceptibility Loci Candidates V. Moreno1 , X. Sole1 , D. Cordero1 , M. Crous-Bou1 , A. Berenguer1 , R. Sanz-Pamplona1 , C. Ruiz-Ponte2 , A. Carracedo2 , S. Castellvi-Bel3 , A. Castells3 . 1 Catalan Institute of Oncology, Cancer Prevention Program, Hospitalet del Llobregat, Spain, 2 University of Santiago de Compostela, Galician Public Foundation of Genomic Medicine, Santiago de Compostela, Spain, 3 Hospital Clinic, Department of Gastroenterology, Barcelona, Spain Background: The identification of genetic susceptibility loci in colorectal cancer though Genome-Wide Association Studies (GWAS) has yielded so far a few hits, but most of the heritability remains to be explained. The identified loci have small effects and, generally, no idea of the carcinogenic mechanisms behind the loci have been proposed. GWAS analysis provide large lists of candidate SNPs but only a few, generally the most significant, are tested in validation studies. Here we hypothesize that SNPs associated to gene expression in normal tissue (eQTLs) have a higher prior probability of being involved in the carcinogenic mechanism. If true, SNPs that are eQTLs should show more significant p-values than expected in a GWAS. Material and Methods: Normal mucosa from 100 patients with colon cancer and 50 healthy donors that underwent colonoscopy have been analyzed with Affimetrix arrays U219 for gene expression and Human Genome 6.0 for genetic variation. To reduce spurious associations, filters were applied beyond quality standards (missing values <10%). Expression probes with low variability (sd <0.26) or low expression (m <2.4) were excluded. SNPs with HWE p < 0.0001 or MAF <0.1 were also excluded. A total of 559,156 SNPs remained for analysis. QTLs were analyzed assuming a linear model on expression, adjusted for cancer status, age and sex. The significance level used for cis associations (SNPs within 1 MB of the target gene) was 1e-4 and for trans association 1e-7. Associations that showed heterogeneity between cases and controls were excluded. The R package MatrixEQTL was used for these analysis. GWAS data was available from the EPICOLON study, a casecontrol study that included 848 cases of colorectal cancer and 629 controls recruited in Spain. Affymetrix Human Genome 6.0 had also been used in this study. The trend test was used to summarize the association of each SNP with colorectal cancer. Results: More than 23,000 SNP-gene associations were identified in cis (3,161 genes) and over 3,700 more in trans (2,275 genes). Though the expression of this large number of genes was modified by SNPs in their genomic region, our hypothesis has not been verified. The distribution of p-values was the same for the eQTL than for the remaining SNPs. Conclusions: Though eQTLs modify the expression of the gene, this effect doesn’t seem to have a relevant impact in the likelihood of these SNPs being susceptibility loci. A more detailed analysis will be presented, studying specifically cancer genes and the distribution of eQTLs in cancer pathways. 1173 Expression of LASS/CerS 4 and 5 in Colon and Endometrial Cancers Z. Dlamini1 , M. Mojakgomo1 , Z. Mbita1 . 1 University of South Africa, Agriculture and Environmental Sciences, Johannesburg, South Africa Introduction: LASS/CerS genes belong to a family of six related genes (CerS1−6). CerS gene products have been shown to produce ceramide, hence their name CerS. Ceramide has been implicated in cancer and apoptosis. A proper regulation of the balance between cell growth and cell death is essential for cellular homeostasis. The precise roles of CerSes in different cancers is not fully understood, especially the role of CerS4 and CerS5 in colon and endometrial cancers. The broad aim of this study was to investigate the role of CerS4 and CerS5 in apoptosis and cancer. Materials and Methods: Bioinformatics tools were used to determine the conservation of these genes in different organisms, and to determine their potential protein-interacting partners, to shed light on their possible roles in cancer. Apoptosis was induced in cultured colon and endometrial cancer cells with their non-cancerous counterparts using 5-FU and Anastrozole respectively. Fluorescence activated cell sorting (FACS) was used to analyse and quantify apoptosis in the untreated and treated cells. Total RNA was then extracted from both treated and untreated cells for cDNA synthesis. The synthesised cDNA was subjected to relative quantitative real-time PCR using LASS4 and LASS5 specific primers. The results were normalised using b-actin. Results and Discussion: LASS genes were shown to be conserved throughout evolution, in different organisms. CerS4 and CerS5 were found to potentially interact with other proteins that may be involved in cell cycle regulation. The cancerous cells showed a higher response to the apoptosis inducing drugs in both colon and endometrial cancers. Using the ANOVA test, FACS analysis results were shown to be statistically significant. Using quantitative real-time PCR, LASS4 and 5 were shown to be up-regulated in both colon and endometrial cancer. However, apoptosis induction resulted in down-regulation of both LASS 4 and LASS 5 in these cancers. Using the Turkey’s test, the results were shown to be statistically significant. These findings implicate the involvement of these genes in cancer and apoptosis.
european journal of cancer 48, suppl. 5 (2012) S25–S288
linkage analysis of the SWR/J × (SWR/J × Car-R) backcross population using high-density marker coverage provided by single nucleotide polymorphism (SNP)-array. Gene expression analysis of normal lung tissues from Car-R and SWR/J was performed; then, we tested the effects of candidate genes (Igfbp2 and Igfbp5) overexpression on clonogenicity of lung cancer lines. Finally, functional analysis of the Igfbp5 promoter alleles from SWR/J and BALB/cJ by luciferase reporter assay was performed. Results and Discussion: We identified the ‘lung and skin tumorigenesis modifier 1’ (Lsktm1) locus on chromosome 1 linked to both skin (LOD score = 3.93) and lung (LOD score = 8.74) tumorigenesis. The Lsktm1 locus was confirmed in a (SWR/J × BALB/cJ)F2 intercross population, indicating that BALB/cJ carries the same cancer resistance alleles of Car-R mouse. Two genes, Igfbp5 and Igfbp2, residing in this locus and belonging to the insulin-like growth factor binding protein family, were expressed at significantly higher levels in normal lung tissue from cancer-resistant Car-R mice than in cancer-susceptible SWR/J mice, and overexpression of the recombinant genes in two lung cancer cell lines significantly inhibited clonogenicity (P < 0.0001). Functional analysis of the Igfbp5 promoter alleles from SWR/J and BALB/cJ by luciferase reporter assay showed differential expression between the strains (P = 0.0001), suggesting a possible involvement of Igfbp5 promoter variants in modulating gene expression. Conclusion: Collectively, our results demonstrate that skin and lung tumors are influenced by a single polymorphic genetic locus and identify Igfbp5 and Igfbp2 as candidate modifier genes of lung tumorigenesis. 1171 DietCompLyf Study − a Multi-centre UK Study on Breast Cancer − What Are the Dietary and Lifestyle Changes Following Diagnosis? K.A. Perkins1 , R. Swann1 , J. Woodside2 , C. Robertson1 , S. Dutton3 , A. Mulligan4 , L. Velentzis5 , M. Keshtgar6 , A. Leathem7 , M. Dwek1 . 1 University of Westminster, Department of Molecular and Applied Biosciences, London, United Kingdom, 2 Queens University, Belfast, United Kingdom, 3 University of Oxford, Centre for Statistics in Medicine, Oxford, United Kingdom, 4 University of Cambridge, Institute of Public Health, Cambridge, United Kingdom, 5 Cancer Epidemiology Research Unit, Cancer Council New South Wales, Sydney, Australia, 6 Royal Free Hospital, Department of Surgery, London, United Kingdom Background: Evidence summarising the influences of dietary and lifestyle factors in prevention of breast cancer is now established, altering the focus of research toward learning the independent and combined effects of features including alcohol, polyunsaturated fat intakes and exercise regimes on breast cancer outcomes. Method: The DietCompLyf study recruited 3,390 female breast cancer patients from 56 NHS centres across the UK. Blood and urine samples were collected alongside information on lifestyle, diet, psychological well-being and quality of life annually for 5 years. Describing the DietCompLyf cohort using tumour biology and treatment profiles, participants’ geographical location, age and ethnicity, the demographics of this cohort are compared with UK breast cancer rates. Additional features specific to the cohort including the socioeconomic status, general health and alcohol consumption are considered alongside changes made to the diet and lifestyle since diagnosis. Multivariate analysis (multiple linear regression) on the changes in BMI and its causes was carried out. Results and Discussion: Recruitment was closed in September 2010 and the collection and validation of baseline data completed. 231 records were excluded for data violation. Provisional analyses of socio-demographic data on the final dataset (3159 women, ages 26−75 years) shows that 88% (2786) were recruited from England, 6% from Wales. This compares well with UK cancer registry data for breast cancer (83% and 5.4% respectively). Populations in Scotland (4.2%) and Northern Ireland (1.4%) were slightly under-represented. In this cohort, 68% live in urban environments, 90% are white, 77% mothers, 70% current alcohol drinkers, 9% smokers and 65% partake in exercise once a week or more at recruitment. The median BMI of this UK cohort is 25.5 kg/m2. Following diagnosis, BMI increased on average by 0.65 kg/m2 . The results of a multivariate analysis on post diagnosis changes in BMI alongside changes in diet, smoking, drinking and exercise habits − including the observations that only 6% of women who drink alcohol at diagnosis and 8% of women who were smoking at diagnosis subsequently give up, will be presented. Conclusion: DietCompLyf is a large UK cohort of breast cancer survivors, the diversity of which is presented here. This cohort provides the opportunity to look at the modifiable changes made by women following a diagnosis of breast cancer. Recurrence and mortality data on members of the cohort being collected will be analyzed with respect to modifiable lifestyle habits once the data are mature. The results will inform advice to patients.
Sunday 8 − Tuesday 10 July 2012
1172 Colorectal Cancer eQTLs as Susceptibility Loci Candidates V. Moreno1 , X. Sole1 , D. Cordero1 , M. Crous-Bou1 , A. Berenguer1 , R. Sanz-Pamplona1 , C. Ruiz-Ponte2 , A. Carracedo2 , S. Castellvi-Bel3 , A. Castells3 . 1 Catalan Institute of Oncology, Cancer Prevention Program, Hospitalet del Llobregat, Spain, 2 University of Santiago de Compostela, Galician Public Foundation of Genomic Medicine, Santiago de Compostela, Spain, 3 Hospital Clinic, Department of Gastroenterology, Barcelona, Spain Background: The identification of genetic susceptibility loci in colorectal cancer though Genome-Wide Association Studies (GWAS) has yielded so far a few hits, but most of the heritability remains to be explained. The identified loci have small effects and, generally, no idea of the carcinogenic mechanisms behind the loci have been proposed. GWAS analysis provide large lists of candidate SNPs but only a few, generally the most significant, are tested in validation studies. Here we hypothesize that SNPs associated to gene expression in normal tissue (eQTLs) have a higher prior probability of being involved in the carcinogenic mechanism. If true, SNPs that are eQTLs should show more significant p-values than expected in a GWAS. Material and Methods: Normal mucosa from 100 patients with colon cancer and 50 healthy donors that underwent colonoscopy have been analyzed with Affimetrix arrays U219 for gene expression and Human Genome 6.0 for genetic variation. To reduce spurious associations, filters were applied beyond quality standards (missing values <10%). Expression probes with low variability (sd <0.26) or low expression (m <2.4) were excluded. SNPs with HWE p < 0.0001 or MAF <0.1 were also excluded. A total of 559,156 SNPs remained for analysis. QTLs were analyzed assuming a linear model on expression, adjusted for cancer status, age and sex. The significance level used for cis associations (SNPs within 1 MB of the target gene) was 1e-4 and for trans association 1e-7. Associations that showed heterogeneity between cases and controls were excluded. The R package MatrixEQTL was used for these analysis. GWAS data was available from the EPICOLON study, a casecontrol study that included 848 cases of colorectal cancer and 629 controls recruited in Spain. Affymetrix Human Genome 6.0 had also been used in this study. The trend test was used to summarize the association of each SNP with colorectal cancer. Results: More than 23,000 SNP-gene associations were identified in cis (3,161 genes) and over 3,700 more in trans (2,275 genes). Though the expression of this large number of genes was modified by SNPs in their genomic region, our hypothesis has not been verified. The distribution of p-values was the same for the eQTL than for the remaining SNPs. Conclusions: Though eQTLs modify the expression of the gene, this effect doesn’t seem to have a relevant impact in the likelihood of these SNPs being susceptibility loci. A more detailed analysis will be presented, studying specifically cancer genes and the distribution of eQTLs in cancer pathways. 1173 Expression of LASS/CerS 4 and 5 in Colon and Endometrial Cancers Z. Dlamini1 , M. Mojakgomo1 , Z. Mbita1 . 1 University of South Africa, Agriculture and Environmental Sciences, Johannesburg, South Africa Introduction: LASS/CerS genes belong to a family of six related genes (CerS1−6). CerS gene products have been shown to produce ceramide, hence their name CerS. Ceramide has been implicated in cancer and apoptosis. A proper regulation of the balance between cell growth and cell death is essential for cellular homeostasis. The precise roles of CerSes in different cancers is not fully understood, especially the role of CerS4 and CerS5 in colon and endometrial cancers. The broad aim of this study was to investigate the role of CerS4 and CerS5 in apoptosis and cancer. Materials and Methods: Bioinformatics tools were used to determine the conservation of these genes in different organisms, and to determine their potential protein-interacting partners, to shed light on their possible roles in cancer. Apoptosis was induced in cultured colon and endometrial cancer cells with their non-cancerous counterparts using 5-FU and Anastrozole respectively. Fluorescence activated cell sorting (FACS) was used to analyse and quantify apoptosis in the untreated and treated cells. Total RNA was then extracted from both treated and untreated cells for cDNA synthesis. The synthesised cDNA was subjected to relative quantitative real-time PCR using LASS4 and LASS5 specific primers. The results were normalised using b-actin. Results and Discussion: LASS genes were shown to be conserved throughout evolution, in different organisms. CerS4 and CerS5 were found to potentially interact with other proteins that may be involved in cell cycle regulation. The cancerous cells showed a higher response to the apoptosis inducing drugs in both colon and endometrial cancers. Using the ANOVA test, FACS analysis results were shown to be statistically significant. Using quantitative real-time PCR, LASS4 and 5 were shown to be up-regulated in both colon and endometrial cancer. However, apoptosis induction resulted in down-regulation of both LASS 4 and LASS 5 in these cancers. Using the Turkey’s test, the results were shown to be statistically significant. These findings implicate the involvement of these genes in cancer and apoptosis.