Allele Loss and Reduced Expression of CYCLOPS Genes is a Characteristic Feature of Chromophobe Renal Cell Carcinoma

Abstract

Copy-number alterations Yielding Cancer Liabilities Owing to Partial losS (CYCLOPS) genes have been recently identified as the most enriched class of copy-number associated gene dependencies in human cancer. These genes are cell essential and render tumor cells highly sensitive to the expression of the remaining copy. Chromophobe renal cell carcinoma (chRCC) is characterized by frequent chromosomal deletions, but the relevance of CYCLOPS genes in this tumor subtype is unclear. We found 39 (31%) of 124 recently published candidate CYCLOPS genes (B. Paolella et al., eLife 2017;6:e23268) located on 7 autosomes that are frequently lost in chRCC. GISTIC and RNA-seq data obtained from the TCGA-KICH database showed that 62% of these CYCLOPS genes had significantly lower expression levels in samples with deletion of the respective gene. As copy number (CN) loss of the CYCLOPS gene SF3B1 (Splicing factor 3B subunit 1) has been recently reported in 71% chRCC, we explored the relevance of SF3B1 CN alteration and SF3B1 expression in a set of chRCC and additional oncocytic renal neoplasms. The frequency of SF3B1 CN loss (65%) was similar to that obtained from the TCGA-KICH database and correlated significantly with both lower SF3B1 mRNA ( P < .05) and protein expression ( P < .001). Other tumor subtypes with oncocytic cytoplasm had normal SF3B1 CN and displayed strong SF3B1 protein expression. These results suggest that CN loss of CYCLOPS genes is a characteristic feature in chRCC. Since many CYCLOPS genes code for components of proteasomes and transcriptional regulation, their alteration could make chRCC vulnerable to targeted drugs.

Loss of CDKN1A mRNA and Protein Expression Are Independent Predictors of Poor Outcome in Chromophobe Renal Cell Carcinoma Patients

“…The corresponding clinical information of TCGA-KICH was obtained from the TCGA Data Portal [57]. Publically available Level 3 TCGA datasets comprising 66 primary chRCCs (TCGA-KICH) were downloaded from the Broad Institute TCGA Genome Data Analysis Center via FIREHOSE [24] including GISTIC copy number (CN) data, Next Generation Sequencing (NGS)-based whole genome sequencing data and RNA-sequencing data as previously described [14][15][16]58]. Two patients with missing or too short follow-up (less than 30 days) were excluded from the Cox regression analysis.…”

“…Punches were taken from tumor areas displaying >90% cancer cells which were marked previously on Hematoxylin and Eosin stained slides. DNA extraction from FFPE tissue was done as previously described [14,15,60]. The double-strand DNA (dsDNA) was quantified by the fluorescence-based Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to manufacturer's instructions.…”

“…Thirty chRCCs had sufficient DNA quality for copy number analysis. Genome-wide DNA copy-number alterations were analyzed by Affymetrix OncoScan ® CNV FFPE Microarrays (Affymetrix, Santa Clara, CA, USA) as previously described [14,15,61]. The samples were processed by IMGM Laboratories GmbH (Martinsried, Germany).…”

“…and H.M.) blinded to the clinical data. The criteria for protein expression analysis were as described in previous TMA studies [15,29]. A tumor was considered CDKN1A positive if ≥ 2% of the tumor cells showed unequivocal nuclear expression.…”

“…Therefore, chRCCs are currently not graded. Interestingly, only recently, it was reported that classic chRCC harbors a larger number of chromosomal losses than in the eosinophilic subtype [14], which is often accompanied by reduced expression of "CYCLOPS" (Copy-number alterations Yielding Cancer Liabilities Owing to Partial losS)" genes [15].…”