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Leveraging population-distinct linkage equilibrium (LD) patterns, trans-ethnic follow-up of variants discovered from genome-wide association studies (GWAS) has proved to be useful in facilitating the identification of bona fide causal variants. We previously developed the preferential LD approach, a novel method that successfully identified causal variants driving the GWAS signals within European-descent populations even when the causal variants were only weakly linked with the GWAS-discovered variants. To evaluate the performance of our approach in a trans-ethnic setting, we applied it to follow up breast cancer GWAS hits identified mostly from populations of European ancestry in African Americans (AA). We evaluated 74 breast cancer GWAS variants in 8,315 AA women from the African American Breast Cancer Epidemiology and Risk (AMBER) consortium. Only 27% of them were associated with breast cancer risk at significance level α=0.05, suggesting race-specificity of the identified breast cancer risk loci. We followed up on those replicated GWAS hits in the AMBER consortium utilizing the preferential LD approach, to search for causal variants or better breast cancer markers from the 1000 Genomes variant catalog. Our approach identified stronger breast cancer markers for 80% of the GWAS hits with at least nominal breast cancer association, and in 81% of these cases, the marker identified was among the top 10 of all 1000 Genomes variants in the corresponding locus. The results support trans-ethnic application of the preferential LD approach in search for candidate causal variants, and may have implications for future genetic research of breast cancer in AA women.

Background: Whole-exome sequencing (WES) has recently emerged as an appealing approach to systematically study coding variants. However, the requirement for a large amount of high-quality DNA poses a barrier that may limit its application in large cancer epidemiologic studies. We evaluated the performance of WES with low input amount and saliva DNA as an alternative source material. Methods: Five breast cancer patients were randomly selected from the Pathways Study. From each patient, four samples, including 3 μg, 1 μg, and 0.2 μg blood DNA and 1 μg saliva DNA, were aliquoted for library preparation using the Agilent SureSelect Kit and sequencing using Illumina HiSeq2500. Quality metrics of sequencing and variant calling, as well as concordance of variant calls from the whole exome and 21 known breast cancer genes, were assessed by input amount and DNA source. Results: There was little difference by input amount or DNA source on the quality of sequencing and variant calling. The concordance rate was about 98% for single-nucleotide variant calls and 83% to 86% for short insertion/deletion calls. For the 21 known breast cancer genes, WES based on low input amount and saliva DNA identified the same set variants in samples from a same patient. Conclusions: Low DNA input amount, as well as saliva DNA, can be used to generate WES data of satisfactory quality. Impact: Our findings support the expansion of WES applications in cancer epidemiologic studies where only low DNA amount or saliva samples are available. Cancer Epidemiol Biomarkers Prev; 24(8); 1207–13. ©2015 AACR.

Genetic and epigenetic alterations have been identified as to contribute directly or indirectly to the generation of transitional cell carcinoma of the urinary bladder (TCC-UB). We have previously found that amplification of chromosome 6p22 is significantly associated with the muscle-invasive rather than superficial TCC-UB. Here, we demonstrated that Sox4, one of the candidate oncogenes located within the chromosome 6p22 amplicon, confers bladder cancer stem cell (CSC) properties. Down-regulation of Sox4 led to the inhibition of cell migration, colony formation as well as mesenchymal-to-epithelial transition (MET). Interestingly, knockdown of Sox4 also reduced the sphere formation, enriched cell population with high levels of aldehyde dehydrogenase (ALDH (high)) and tumor formation potential. Using gene expression profiling, we further identified novel Sox4 target genes. Last, immunohistochemistry analysis of human bladder tumor tissue microarrays (TMAs) indicated that high Sox4 expression was correlated with advanced cancer stages and poor survival rate. In summary, our data show that Sox4 is an important regulator of the bladder CSC properties and it may serve as a biomarker of the aggressive phenotype in bladder cancer.

We screened the whole tumor genome to identify DNA copy number gains and losses that discriminate between primary breast carcinomas (MP) and their nodal metastases (ML). Six candidate genes were confirmed by quantitative PCR to have differentially distributed copy number changes. Three of the genes (ERRγ, DDX6, and TIAM1) were more commonly amplified in nodal metastases. Principal component analysis revealed that MP-ML pairs varied markedly in their genomic divergence. The latter was larger in PR-negative tumors. Nodal metastases may form early or late in the development of breast carcinomas and PR-negative tumors may metastasize earlier or are genomically less stable.

We employed a whole genome tumor profiling approach in an attempt to identify DNA copy number alterations (CNAs) and new candidate genes that are correlated with the metastatic potential of a primary breast carcinoma and with progression at the metastatic site. Fifty-four small (≤2 cm), high grade, ER-positive, formalin-fixed invasive ductal carcinomas were suitable for whole genome profiling analysis. Twenty-four of them did not form metastases within 5–10 years (unmatched primaries, UP). Thirty tumors had at least one synchronous axillary lymph node metastasis (matched primaries, MP; matched lymph node metastases, ML). Genomic DNA was hybridized to high density (19k) BAC arrays. Statistical analysis revealed differential distributions of CNAs between UP and MP and between MP and ML, respectively. We selected 27 candidate genes for validation experiments using quantitative (Q-)PCR of genomic DNA. For tetraspanin TSPAN1, we studied mRNA expression levels in a separate cohort of primary breast carcinomas and in breast cell lines. Matched primary (MP) tumors had a threefold higher rate of DNA copy number losses compared to UP tumors. In the UP-MP comparison, 186 BACs were differentially amplified or deleted. Most of them were localized to chromosomes 7p, 16q and 18q. In the MP-ML comparison, 131 BACs showed differential CNAs. Most of them were localized to chromosomes 1q and 20. By Q-PCR, seven candidate genes could be confirmed to show differential distributions of CNAs. TSPAN1 was amplified in UP and deleted in MP tumors. The gene was markedly downregulated in ER-negative and high-grade breast cancers. Metastasizing tumors had a higher rate of deletions, suggesting possible inactivation of metastasis suppressor genes. We provide preliminary evidence that TSPAN1 may be another important breast cancer suppressor gene belonging to the tetraspanin superfamily.

The goal of this study was to identify recurrent regions of genomic gain or loss in endometrial cancer of the endometrioid type in the context of racial disparities in mortality for this disease. Array comparative genomic hybridization (aCGH) analysis was performed on 80 frozen primary tumors from the Gynecologic Oncology Group (GOG)-210 bank using the RPCI 19K BAC arrays. The 80 patients included 20 African American (AA) Stage I, 20 White (W) Stage I, 20 African American (AA) Stage IIIC/IV, and 20 White (W) Stage IIIC/IV. A separate subset of 220 endometrial cancers with outcome data was used for validation. A 1.6-Mbp region of gain at 1q23 was identified by aCGH in all AA patients and high grade W patients, but not W low grade patients. In the validation arm of 220 patients copy number gain at this region was validated using FISH and locus specific BACs. The number of AA patients in the validation arm was too small to confirm the aCGH association with racial disparity. Kaplan-Meier curves for survival showed a significant difference for gain at 1q23 versus no gain (log rank P = 0.0014). When subdivided into various groups of risk by stage and grade the survival curves showed a decreased survival for high grade and/or stage tumors, but not for low grade and/or stage endometrioid tumors. Univariate analyses for gain at 1q23 showed a significant association (P = 0.009) with survival. Multivariate analysis for gain at 1q23 did not show a significant association with survival (P = 0.14).