Examples of The Cancer Genome Atlas (TCGA) Analyses in GenePool

Transcription

1 Examples of The Cancer Genome Atlas (TCGA) Analyses in 1. RNA-Seq 2. mirna-seq 3. Protein Expression 4. Somatic Mutations 5. Copy Number 6. DNA Methylation RNA-Seq All primary tumor samples (1,097 of 1,218) from the breast invasive carcinoma (BRCA) set. Expression Profile workflow using Gene regions, 75th percentile normalization and Log2 scaling. Select PAM50 GeneList from the Reference Annotations in the Gene menu in Annotation. Unselect All other results to show only the PAM50 genes. Enter Heatmap view with hierarchical clustering. Add one or more dataset annotations from ER Status, PR Status, HER2/neu Status and/or PAM50 (independently characterized) from the new Data Annotations menu under View Details. Note that the dataset annotations are stratified by gene-level expression, as expected, shown in the orange oval: Basal-like

2 mirna-seq All primary tumor samples (775 of 869) from the breast invasive carcinoma (BRCA) set. Expression Comparison workflow of PAM50 classified 353 Luminal A (Group 1) versus 134 Basal using Gene regions, 75th percentile Normalization and Log2 Scale. Select most significantly differentially expression mirnas with p < 1e -6 and view Heatmap. clusters Luminal A and Basal samples with the top differentially expression mirnas: Basal-like TCGA mirna-seq data confirms many of the mirnas previously shown to differentiate tumors classified as Luminal A or Basal in an independent cohort of 101 patients profiled using microarrays: Enerly et al. (2001) mirna-mrna Integrated Reveals Roles for mirnas in Primary Breast Tumors. PLoS ONE 6:(2):e [Figure 1B]

3 Protein Expression All primary tumor samples (410) from the breast invasive carcinoma (BRCA) set. Expression Profile workflow using Gene regions, and Do not normalize. Returns protein expression levels for 142 genes. Enter Heatmap view with hierarchical clustering. Add one or more dataset annotations from ER Status, PR Status, HER2/neu Status and/or PAM50 (independently characterized) from the new Data Annotations menu under View Details. Note that the dataset annotations are clustered by protein expression, shown in the orange oval: Basal-like (ER-, PR-)

4 Somatic Mutations 1,097 breast invasive carcinoma (BRCA) samples with somatic mutations profiles by Washington University Working Group (AWG). Variation Profile workflows for 490 Luminal A, 235 Luminal B, 103 HER2-enriched and 188 Basal-like Primary Tumor samples. For each variation profile, select for High or Moderate impact mutations (i.e. non-silent) Focus on Cancer Gene Census (GCG) genes, if desired and view as a Gene Pivot Note the genes with most frequency non-silent somatic mutations in are similar to those published by TCGA. Note that frequency of p53 somtatic mutations calculated by increases dramatically with expression-classified groups of worsening prognosis (e.g. Luminal A < Luminal B < HER2- enriches < Basal-like) Note similar pattern of frequencies between and publication for the top genes within the four expression-classified groups. Gene Luminal A Luminal B HER2 Basal PIK3CA 44.5% 28.5% 38.8% 5.3% TP53 9.2% 25.1% 66.0% 74.5% MAP3K1 10.8% 5.1% 4.9% 1.6% MAP2K4 4.7% 3.4% 1.0% 0.0% GATA3 11.0% 16.6% 2.9% 1.1% MLL3 8.0% 7.2% 5.8% 4.8% CDH1 17.8% 7.7% 5.8% 0.5% PTEN 3.9% 3.0% 3.9% 2.7% The Cancer Genome Atlas Network (2012) Comprehensive molecular portraits of human breast tumours. Nature 490, [Figure 1]

5 Copy Number All primary tumor samples (1,093 of 2,218) from the breast invasive carcinoma (BRCA) set. Expression Comparison workflow of HER2/nue Status 175 Positive (Group 1) versus 757 Negative (Group 2) using Gene regions, and Do not normalize. Select top differentially amplified genes with fold difference > 1 and p < 1e -6, and then sort by decreasing fold difference. Turn on Chromosome Fraction annotation column. Note that all genes that show amplification at the top of the list, including ERBB2 (HER2/ nue, which is 2nd on the list), are located in the same region on chromosome 17. And note that a significant fraction of HER2+ samples (as determined by IHC or FISH) have amplification of ERBB2 in the bar chart:

6 Methylation All primary tumor and solid tissue normal samples from the breast invasive carcinoma (BRCA) set. Expression Comparison workflow of 778 Primary Tumor (Group 1) versus 98 Solid Tissue Normal (Group 2) using Gene regions, and Do not normalize, do not scale. Select top differentially methylated genes with fold difference > 1.5 and p < 1e -6. Genes reported independently as gaining methylation in 20 breast cancer samples have increased methylation of more than 50% on average in tumor samples as compared to solid tissue normal samples in TCGA ( methylation ratios are shown in orange below, beside the published data) NKX2-1 CCDC140 (3 genes) (3 genes) Wojdacz et al. (2014) Identification and characterization of locus-specific methylation patterns within novel loci undergoing hypermethylation during breast cancer pathogenesis. Breast Cancer Research 16:R17. [Table 1]

7 ! Furthermore, viewing the Enrichment for genes that gain methylation shows enrichment for functions that involved in cancer right at the top: And the gene methylation levels for one gene (POU4F) are significantly higher for many tumor samples: