Next-Generation RNA Sequencing-Based Biomarker Characterization of Chromophobe Renal Cell Carcinoma and Related Oncocytic Neoplasms - Beyond the Abstract

The cell types that line the renal nephronal tubular channels are broadly named by their anatomical location as proximal to distal tubules via the loop of Henle. Histomorphologic and proteogenomic studies to identify and functionally characterize the cell types in the normal state as well as alterations in chronic, acute, and neoplastic diseases are further complemented by standard and high throughput molecular and cytogenetic techniques. Particularly in neoplastic settings, determination of cell-of-origin for renal cell carcinoma (RCC) subtypes will help us understand the process of cellular transformation; importantly this would allow distinguishing the transcriptomic landscape of the tumor epithelia that are shared with benign cell types (lineage/cell type-specific markers) from tumor-specific events, thereby enabling identification of novel tumor biomarkers. While several groups have postulated the cell of origin of the diverse array of kidney neoplasms, the precise knowledge on the types of target cell that undergo neoplastic transformation within the kidney ultimately resulting in an incredible array of morphological presentations under the microscope is still limited. Integrative analysis of RNAseq/genomics data now available for several RCC subtypes can serve as a good starting point to explore these concepts.

Our study describes an integrative pan-RCC RNA sequencing data analysis, with our workflow termed “Renaissance”, to primarily identify the RCC subtype-specific transcriptomes. One interesting observation we made early on in this pan-RCC analysis was the ability of FOXI1 and its genomic neighbor LINC01187 gene expression to unequivocally identify all the chromophobe RCC (ChRCC) samples (Figures 1A and 1B), both within our cohort and in The Cancer Genome Atlas “TCGA” RCC cases. Importantly, the ChRCC cases that were misclassified in the initial TCGA study were correctly identified by FOXI1/LINC01187 expression alone. Conversely, in our cohort, where the lack of expression of these genes was seen in a few cases of chRCC (at the histomorphologic level), subsequent genomic characterization identified MTOR mutations instead of the recurrent chromosomal copy loss patterns associated with chRCC. Using immunohistochemistry (IHC) to assess FOXI1 and RHCG proteins and RNA in situ hybridization (RNA-ISH) for LINC01187 mRNA expression, we carried out an exhaustive validation and showed the diagnostic utility of these genes for ChRCC in primary and metastatic settings.

Our study brings these markers a step closer to the clinic, pending validation in independent cohorts by other groups. Interestingly, the signal patterns we observed yielded additional information; for example, the membranous staining patterns of RHCG discerned the spectrum of oncocytic tumors, while the nuclear FOX1 and LINC01187 staining differentiated between classic (high expression) and sarcomatoid ChRCC (loss of expression). The repercussions of the latter observation in sarcomatoid chRCC need to be further explored to identify therapeutic vulnerabilities.

Next, when we examined the expression pattern of FOXI1/LINC01187 in publicly available human normal/tumor tissue RNAseq cohorts, we discovered the remarkable specificity in tissue expression exhibited by these genes. Here, our initial analysis with data from GTEX study and solid tissue tumor/normal pairs from the TCGA (10,000 samples from 33 different cancer types), revealed that the mRNA levels of FOXI1 and LINC01187 genes was lower in normal kidneys and significantly overexpressed in ChRCC tumors. As indicated by our single-cell sequencing data, IHC and RNA-ISH assays, the low-level expression we observed in normal kidneys is derived from renal intercalated cells. This highly restricted and shared expression in the intercalated cells of the distal nephron and ChRCC supports the former as a cell of origin for ChRCC. Similar investigational studies are bound to enhance our understanding of the molecular underpinnings and associated cell of origin and biomarker assessments for other renal tumors.

high level LINC01187 expression figure A

Figure 1A
. RNA in situ hybridization demonstrates high-level LINC01187 expression in ChRCC 

high level LINC01187 expression figure B

Figure 1B
. RNA in situ hybridization demonstrates high-level LINC01187 expression

Written by: Rohit Mehra, MD, Associate Professor, Genitourinary Pathology, Member, Michigan Center for Translational Pathology (MCTP), Director, MCTP Esoteric Clinical Laboratory Services, MLabs Genitourinary Service Line Director, Director, Michigan Legacy Tissue Program (MLTP), Department of Pathology, Michigan Medicine, University of Michigan, Ann Arbor, Michigan; Saravana Dhanasekaran, PhD, Associate Research Scientist, Department of Pathology, Michigan Medicine, University of Michigan, Ann Arbor, Michigan

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