Intrinsic Genomic Differences Between African American and White Patients With Clear Cell Renal Cell Carcinoma

IMPORTANCE There are well-documented racial disparities in outcomes for African American patients with clear cell renal cell carcinoma (ccRCC). Despite a dramatic improvement in overall survival in white patients since the advent of targeted therapy, survival for African Americans with advanced ccRCC has not changed. There is little known about potential racial differences in tumor biology of ccRCC. OBJECTIVE To determine if there are racial differences in the somatic mutation rate and gene expression of ccRCC tumors from white and African American patients. DESIGN, SETTING, AND PARTICIPANTS Overall, 438 patients with ccRCC were identified through The Cancer Genome Atlas (TCGA) clear cell kidney (KIRC) dataset (419 white and 19 African American patients). The GSE25540 dataset containing 135 patients (125 white and 10 African American patients) was used for validation. Tumor samples were collected from numerous cancer centers and were examined for racial differences in somatic mutation rates and RNA expression. Racial differences in somatic mutation rates and RNA expression were examined.


R
enal cell carcinoma (RCC) is the eighth most commonly diagnosed cancer in the United States.3][4] Despite this documented survival disparity, there is no data examining genomic or transcriptomic differences of clear cell RCC (ccRCC) in African American patients vs white patients.
The last decade has produced a wealth of knowledge about the molecular drivers of RCC, including the comprehensive molecular characterization of ccRCC by The Cancer Genome Atlas (TCGA). 5A hallmark event in the development of ccRCC is loss of heterozygosity of the von Hippel-Lindau (VHL) tumor suppressor gene.VHL inactivation occurs at a rate of 52% to 82% in sporadic ccRCC 5 and leads to stabilization of the α subunit of the hypoxia-inducible factor (HIF) family of transcription factors.Stabilization of HIFα subunits results in their heterodimerization with HIFβ subunits and a transcriptionally active complex whose target genes are intimately involved in tumor angiogenesis.The vascular endothelial growth factor (VEGF) pathway in particular is a key transcriptional HIF target and is targeted by a number of the currently US Food and Drug Administration-approved therapies.In addition, several other genes, such as PBRM1, SETD2, and BAP1, are also mutated in ccRCC patient tumors. 6Herein, we investigate potential genetic differences in ccRCC that are associated with race.

Methods
The Cancer Genome Atlas and Validation Data and Analysis RNA sequencing, somatic mutation, and copy number data from the TCGA kidney clear cell (KIRC) data set were downloaded from the publically available TCGA data portal (https: //tcga-data.nci.nih.gov/tcga/), and somatic mutation data was downloaded from an independent and publicly available data set from Peña-Llopis et al 6

(GSE25540 data set).
The TCGA RNaseq data set was log2 transformed and median-centered.Two-class significance analysis of microarrays (using 2-fold change and false discovery rate = 0) was performed to generate race-specific gene lists. 7The significant genes and corresponding fold changes as determined by significance analysis of microarrays were analyzed by Ingenuity Pathway Analysis (Ingenuity Systems) for predicted pathway activation and/or inhibition.Gene Set Enrichment Analysis (Broad Institute) was performed comparing ccRCC tumors from African American patients vs white patients (ethnicity defined by the TCGA) against Molecular Signature Database c2.all.v4.0.The mutations in the 9 most commonly mutated genes-VHL, PBRM1, SETD2, KDM5C, PTEN, BAP1, MTOR, TP53, and PI3KCA 7 -were analyzed between African American patients and white patients.Patients were then classified into the previously described RNA subtypes of ccRCC-ccA and ccB-based on patterns of differential gene expression using prediction analysis of microarray. 8,9

Statistical Analysis
Differences in somatic mutation rates by race were compared using χ 2 or Fisher exact test where applicable.Fisher exact test was used to compare prevalence of the ccA and ccB molecular subtypes between races.

Results
We first investigated whether there are racial differences in the mutational spectrum of ccRCC tumors comparing data from African American patients (n = 19) and white patients (n = 419) from the TCGA ccRCC KIRC data set.Overall, 175 of 351 (50%) white patients had VHL mutations, whereas 2 of 12 (17%) African American patients had VHL mutations (P = .04)(Figure 1A) (eTable 1 in the Supplement).In contrast, there were no racial differences in the mutational frequency of other TCGA KIRC-defined significantly mutated genes (Figure 1A) (eTable 1 in the Supplement).These results were validated for VHL, PBRM1 and BAP1 in an independent data set verifying the lower prevalence of VHL mutations in African American patients (Figure 1B) (eTable 1 in the Supplement) 6 .
Given the lower frequency of VHL mutations in African American patients, we investigated the RNA expression of the VEGF ligands and VEGF receptors to evaluate for downstream effects of VHL loss.In the TCGA KIRC data set, 419 white patients and 19 African American patients were available for analysis.In the TCGA data set, African American patients had significantly lower expression of the VEGFA ligand (eFigure 1A in the Supplement), as well as the FLT-1 (VEGFR1) and KDR (VEGFR2) receptors (eFigure 1B in the Supplement).Furthermore, significance analysis of microarrays and subsequent ingenuity pathway analysis of upstream regulators predicted that numerous genes associated with HIF (EPAS1, HIF1A, ARNT, and CREB1) transcriptional regulation were up-regulated in white patients (eTable 2 in the Supplement).Finally, several VEGF and HIF signatures were negatively enriched in African American patients as determined by Gene Signature Enrichment Analysis (eFigure 3 and eTable 3 in the Supplement).In

Key Points
Question Are there genomic differences between clear cell renal cell carcinomas (ccRCC) that arise in African American patients and white patients that may contribute to the disparity in survival outcomes observed between races?Findings Using data from the The Cancer Genome Atlas clear cell kidney project, we found that African American patients have a significantly lower rate of mutations of the von Hippel-Lindau tumor suppressor gene, as well as decreased hypoxia-inducible factor activation.In addition, African American patients are enriched for a previously defined RNA subtype (ccB) that is associated with worse survival outcomes.
Meaning There are distinct racial differences in the biology of ccRCC tumors that may partially explain the well-documented racial disparity in survival outcomes of African American patients with advanced ccRCC.

Discussion
This is the first report to knowledge to examine genomic differences between ccRCC in African American patients vs white patients.Our results indicate that ccRCC tumors from African American patients exhibit a lower rate of VHL mutation and a correspondingly lower level of HIF and VEGF pathway activation.We therefore postulate that a significantly larger proportion of tumors from African American p at i e nt s m ay h ave a H I F-i n d e p e n d e nt a n d V E G F-

Figure 1 .
Figure 1.Gene Mutations in Clear Cell Renal Cell Carcinoma Tumors by Data Set Source and Race

Figure 2 .
Figure 2. Incidence and Correlation to Centroid of Clear Cell Renal Cell Carcinoma Tumor Subtype