Next-generation RNA Sequencing-based Biomarker Characterization of Chromophobe Renal Cell Carcinoma and Related Oncocytic Neoplasms

Abstract

Background: Renal cell carcinomas (RCCs) are a heterogeneous group of neoplasms. Recent sequencing studies revealed various molecular features associated with histologic RCC subtypes, including chromophobe renal cell carcinoma (ChRCC).

Objective: To characterize the gene expression and biomarker signatures associated with ChRCC.

Design, setting, and participants: We performed integrative analysis on RNA sequencing data available from 1049 RCC specimens from The Cancer Genome Atlas and in-house studies. Our workflow identified genes relatively enriched in ChRCC, including Forkhead box I1 (FOXI1), Rh family C glycoprotein (RHCG), and LINC01187. We assessed the expression pattern of FOXI1 and RHCG protein by immunohistochemistry (IHC) and LINC01187 mRNA by RNA in situ hybridization (RNA-ISH) in whole tissue sections representing a cohort of 197 RCC cases, including both primary and metastatic tumors.

Outcome measurements and statistical analysis: The FOXI1 and RHCG IHC staining, as well as the LINC01187 RNA-ISH staining, was evaluated in each case for intensity, pattern, and localization of expression.

Results and limitations: All primary and metastatic classic ChRCCs demonstrated homogeneous positive labeling for FOXI1, RHCG proteins, and LINC01187 transcript. Unclassified RCC with oncocytic features, oncocytoma, and hybrid oncocytic tumor, as well as all but two cases of eosinophilic ChRCC also stained positive. Importantly, metastatic and primary RCC of all other subtypes did not demonstrate any unequivocal staining for FOXI1, RHCG, or LINC01187. In normal kidney, FOXI1, RHCG, and LINC01187 were detected in the distal nephron segment, specifically in intercalated cells. Two cases of eosinophilic ChRCC with focal expression of FOXI1 and LINC01187, and Golgi-like RHCG staining were found to contain MTOR gene mutations upon DNA sequencing.

Conclusions: We demonstrate a pipeline for the identification and validation of RCC subtype-specific biomarkers that can aid in the confirmation of cell of origin and may facilitate accurate classification and diagnosis of renal tumors.

Patient summary: FOXI1, RHCG, and LINC01187 are lineage-specific signature genes for chromophobe renal cell carcinoma.

Keywords: Chromophobe; Classification; Hybrid oncocytic tumor; Immunohistochemistry; Next-generation sequencing; RNA in situ hybridization; Renal cell carcinoma.

Figure 1.

Nomination of cancer and lineage biomarkers of ChRCC. A) Schematic of the gene analysis pipeline to determine cancer specific and lineage specific expression in ChRCC. B) Volcano plot represents significantly differentially expressed genes in ChRCC compared to normal and other RCC subtypes. Selected top up- and down-regulated genes are circled and labeled. C) Expression of top ChRCC lineage specific genes across major RCC subtypes. KICH = ChRCC, KIRC = CCRCC, KIRP = PRCC. D) Expression of top ChRCC lineage specific genes across major epithelial cell types in human kidney (from single cell sequencing). PT = proximal tubule, LOH = loop of Henle, DT = distal tubule, PC = principle cell, IC = intercalated cell. E) Expression of top ChRCC cancer specific genes across major RCC subtypes. F) Nominated biomarkers are highly specific to ChRCC. Initial and final diagnoses (after re-evaluation) of TCGA RCC cases are annotated on the top of the heatmap. Cancer samples with high expression of nominated biomarkers were almost exclusively ChRCC, including cases with confusing histology (i.e., cases were re-assigned to different subtypes).

Figure 2.

Biomarker Expression in Normal Kidney. Normal kidney tissue (A, H&E, 200x) demonstrates nuclear staining for FOXI1 (B, 200x), circumferential membranous staining for RHCG (C, 200x), and high level nuclear expression of LINC01187 (D, 200x) in distal tubules. Scale bar = 100 microns.

Figure 3.

Classic Chromophobe Renal Cell Carcinoma (ChRCC). Primary (A, H&E, 200x) and metastatic (B, H&E, 200x) ChRCC demonstrate strong nuclear staining for FOXI1 (C and D, 200x), circumferential membranous staining for RHCG (E and F, 200x), and high level nuclear LINC01187 expression (G and H, 200x). Scale bar = 100 microns.

Figure 4.

Clear Cell Renal Cell Carcinoma (CCRCC). Primary (A, H&E, 200x) and metastatic (B, H&E, 200x) CCRCC are negative for FOXI1 (C and D, 200x), RHCG (E and F, 200x), and LINC01187 (G and H, 200x) expression. Scale bar = 100 microns.

Figure 5.

Dual RNA in situ Hybridization for FOXI1 and LINC01187. Co-expression of FOXI1 (Red) and LINC01187 (Green) was seen in the majority of classic ChRCC tumor cells (A, 200x and B, 400x), as well as in the intercalated cells in normal kidney tissue (C, 200x and D, 400x). Scale bar = 100 microns for A and C, 50 microns for B and D.

Figure 6.

Biomarker Expression in Metastatic ChRCC. Positive expression of FOXI1, RHCG, and LINC01187 by metastatic ChRCC at four different anatomic sites, including omentum, right psoas muscle, stomach, and retroperitoneal lymph nodes. (A, H&E, 100x; B, FOXI1 IHC 100x; C, RHCG IHC 100x; D, LINC01187 RNAISH 100x)