Final Report Summary - CANCERGENE (Identification and functional characterization of genetic cancer risk variants)
Project objectives
Genome-wide association studies (GWAS) have led to the discovery of single nucleotide polymorphisms (SNPs) that associate with risk of various types of cancer. However, the causative genetic variation at the corresponding loci has in very few cases been characterise d. The major objectives of CANCERGENE were:
1) to discover cancer causing polymorphisms;
2) functionally validate the newly identified variants in human cells;
3) to throw light on biological properties associated with prostate cancer risk alleles located at 8q24.
Work performed and summary of results
Objective 1:
In order to identify sequence variants that associate with risk of cancer, GWAS were performed on several cancer types, including cancers of the prostate, breast, urinary bladder, lung, ovary and skin, using Icelandic cancer cases and controls. Follow-up of selected variants in independent case-control sample sets of European descent led to the discovery of eight variants that associate with cancer (four for prostate cancer, two for bladder cancer, one for lung cancer and one for ovarian cancer). Variants at six loci that associate with levels of prostate specific antigen (PSA) were identified but these variants may be utilised to improve the PSA screening test for prostate cancer. In addition, two variants were identified that affect the risk of breast cancer and basal cell carcinoma of the skin, respectively, but only when the variant is inherited from the father. Finally, the Icelandic bladder cancer cases and controls were used to help validate several variants that had been found by external investigators to associate with risk of bladder cancer. Fine-mapping of the association signals was attempted using bioinformatics approaches and additional genotyping for each locus. Using this approach, it was possible to refine a previously published association signal for breast cancer at the oestrogen receptor locus. Finally, 27 non-synonymous variants in cancer-associated genes that had been found by sequencing were tested for association to cancer in an Icelandic sample set of 5000 cancer cases and 30 000 controls. Association to cancer was found for two SNPs, however, these associations did not replicate in external cohorts.
Objective 2:
The functional validation of newly identified variants was performed using several approaches. Firstly, isogenic cell lines, discordant only for a lung cancer risk variant in the nicotinic acetylcholine receptor gene, CHRNA5, were produced but functional characterisation of these cells has not been completed. Studies on tumour tissues and cell lines were performed on several cancer risk variants identified through GWAS and correlation with clinical parameters was also investigated. Notably, for the breast cancer variant at 11p15 that shows a parent-of-origin effect, we identified a differentially methylated CTCF-binding site in the region and demonstrated correlation between the variant and decreased methylation. Also, a bladder cancer variant at the FGFR3 locus shows a stronger association with tumours that have a low risk of progression than with tumours that have a higher risk of progression. Notably, the variant also associates with increased expression of FGFR3 and TACC3 in adipose tissue and FGFR3 protein level and somatic mutations of the FGFR3 gene in Ta bladder tumours.
Objective 3:
In order to test if the prostate cancer risk variants at 8q24 are located in functional sites, we performed a number of studies. Transcripts in the 8q24 region were mapped by hybridising RNA from prostate tissues to a tiled microarray covering the risk region, ChIP studies were performed to identify transcription initiation sites, ChIP-Seq studies were conducted to identify methylated sites in the region and genomic DNA from blood was hybridised to the microarray chip to characterise germ-line copy number variants in the region. To summarise , no association was found between cancer risk variants or any of the following:
1) expression of transcripts;
2) transcript initiation sites;
3) methylation sites; or
4) copy number variation.
In parallel studies, copy number at 8q24 was evaluated by fluorescence in situ hybridisation (FISH) in prostate tumours positive or negative for two prostate cancer risk variants. The mean copy number in tumours from carriers and non-carriers was similar indicating absence of selection for risk alleles. Paraffin embedded prostate tumour samples and cell lines were screened for genomic rearrangement in the 8q24 region. No somatic genomic rearrangements were identified that correlated with 8q24 risk genotypes. The genotype was not correlated with TMPRSS2-ERG fusions on chromosome 21.
The results from studies funded by CANCERGENE have been reported in nine peer-reviewed publications and the IP protection has been sought for the genetic findings.
Potential impact and use of results
Genome-wide association studies opened a new dimension in cancer genetics, resulting in the discovery of numerous variants that associate with the disease. The first generation of genetic risk assessment tests based on these findings are already in the market, but the utility of these tests will grow rapidly as more variants are uncovered and included. One of the major goals of CANCERGENE was to identify new cancer risk variants which can be used to produce additional tests and increase the utility of existing tests. The discovery of variants that associate with PSA levels is potentially also very important. The PSA screening test is widely used for detection of prostate cancer but its use has been controversial because of the lack of specificity and sensitivity. Estimates of the effect of genetic variation on PSA levels, as well as on prostate cancer risk, could lay a foundation for the development of individual prostate cancer screening strategies that would have the ultimate goal of reducing cost and improving quality of life.
The genetic risk variants identified to date tag an association between a genetic region and cancer but are unlikely to be the actual causative variants. In order to elucidate the actual causative variation, fine-mapping and functional studies on each locus were performed. Using tumour studies, functional assays and chromatin analysis, we attempted to increase our knowledge about the biological pathways that play a role in cancer initiation and progression. For several risk loci, our studies provided new insights into the genetic underpinnings of cancer risk. This knowledge may in the future be used to help develop more effective prevention and treatment strategies.
CANCERGENE website http://www.cancer-gene.eu
Genome-wide association studies (GWAS) have led to the discovery of single nucleotide polymorphisms (SNPs) that associate with risk of various types of cancer. However, the causative genetic variation at the corresponding loci has in very few cases been characterise d. The major objectives of CANCERGENE were:
1) to discover cancer causing polymorphisms;
2) functionally validate the newly identified variants in human cells;
3) to throw light on biological properties associated with prostate cancer risk alleles located at 8q24.
Work performed and summary of results
Objective 1:
In order to identify sequence variants that associate with risk of cancer, GWAS were performed on several cancer types, including cancers of the prostate, breast, urinary bladder, lung, ovary and skin, using Icelandic cancer cases and controls. Follow-up of selected variants in independent case-control sample sets of European descent led to the discovery of eight variants that associate with cancer (four for prostate cancer, two for bladder cancer, one for lung cancer and one for ovarian cancer). Variants at six loci that associate with levels of prostate specific antigen (PSA) were identified but these variants may be utilised to improve the PSA screening test for prostate cancer. In addition, two variants were identified that affect the risk of breast cancer and basal cell carcinoma of the skin, respectively, but only when the variant is inherited from the father. Finally, the Icelandic bladder cancer cases and controls were used to help validate several variants that had been found by external investigators to associate with risk of bladder cancer. Fine-mapping of the association signals was attempted using bioinformatics approaches and additional genotyping for each locus. Using this approach, it was possible to refine a previously published association signal for breast cancer at the oestrogen receptor locus. Finally, 27 non-synonymous variants in cancer-associated genes that had been found by sequencing were tested for association to cancer in an Icelandic sample set of 5000 cancer cases and 30 000 controls. Association to cancer was found for two SNPs, however, these associations did not replicate in external cohorts.
Objective 2:
The functional validation of newly identified variants was performed using several approaches. Firstly, isogenic cell lines, discordant only for a lung cancer risk variant in the nicotinic acetylcholine receptor gene, CHRNA5, were produced but functional characterisation of these cells has not been completed. Studies on tumour tissues and cell lines were performed on several cancer risk variants identified through GWAS and correlation with clinical parameters was also investigated. Notably, for the breast cancer variant at 11p15 that shows a parent-of-origin effect, we identified a differentially methylated CTCF-binding site in the region and demonstrated correlation between the variant and decreased methylation. Also, a bladder cancer variant at the FGFR3 locus shows a stronger association with tumours that have a low risk of progression than with tumours that have a higher risk of progression. Notably, the variant also associates with increased expression of FGFR3 and TACC3 in adipose tissue and FGFR3 protein level and somatic mutations of the FGFR3 gene in Ta bladder tumours.
Objective 3:
In order to test if the prostate cancer risk variants at 8q24 are located in functional sites, we performed a number of studies. Transcripts in the 8q24 region were mapped by hybridising RNA from prostate tissues to a tiled microarray covering the risk region, ChIP studies were performed to identify transcription initiation sites, ChIP-Seq studies were conducted to identify methylated sites in the region and genomic DNA from blood was hybridised to the microarray chip to characterise germ-line copy number variants in the region. To summarise , no association was found between cancer risk variants or any of the following:
1) expression of transcripts;
2) transcript initiation sites;
3) methylation sites; or
4) copy number variation.
In parallel studies, copy number at 8q24 was evaluated by fluorescence in situ hybridisation (FISH) in prostate tumours positive or negative for two prostate cancer risk variants. The mean copy number in tumours from carriers and non-carriers was similar indicating absence of selection for risk alleles. Paraffin embedded prostate tumour samples and cell lines were screened for genomic rearrangement in the 8q24 region. No somatic genomic rearrangements were identified that correlated with 8q24 risk genotypes. The genotype was not correlated with TMPRSS2-ERG fusions on chromosome 21.
The results from studies funded by CANCERGENE have been reported in nine peer-reviewed publications and the IP protection has been sought for the genetic findings.
Potential impact and use of results
Genome-wide association studies opened a new dimension in cancer genetics, resulting in the discovery of numerous variants that associate with the disease. The first generation of genetic risk assessment tests based on these findings are already in the market, but the utility of these tests will grow rapidly as more variants are uncovered and included. One of the major goals of CANCERGENE was to identify new cancer risk variants which can be used to produce additional tests and increase the utility of existing tests. The discovery of variants that associate with PSA levels is potentially also very important. The PSA screening test is widely used for detection of prostate cancer but its use has been controversial because of the lack of specificity and sensitivity. Estimates of the effect of genetic variation on PSA levels, as well as on prostate cancer risk, could lay a foundation for the development of individual prostate cancer screening strategies that would have the ultimate goal of reducing cost and improving quality of life.
The genetic risk variants identified to date tag an association between a genetic region and cancer but are unlikely to be the actual causative variants. In order to elucidate the actual causative variation, fine-mapping and functional studies on each locus were performed. Using tumour studies, functional assays and chromatin analysis, we attempted to increase our knowledge about the biological pathways that play a role in cancer initiation and progression. For several risk loci, our studies provided new insights into the genetic underpinnings of cancer risk. This knowledge may in the future be used to help develop more effective prevention and treatment strategies.
CANCERGENE website http://www.cancer-gene.eu