The clinical classification of renal tumors is being continuously refined based on the advances in morphological and molecular characterizations of novel and established renal tumor entities. Solid tumor molecular characterization efforts lead by various consortiums (CPTAC, TCGA) and select groups such as ours, have utilized next-generation sequencing and other high-throughput technologies to discover subtype-specific renal tumor molecular aberrations and biomarkers.
In this study, we presented our discovery and validation of the TRIM63 gene, which encodes an E3 ubiquitin ligase, as a specific biomarker of microphthalmia-associated transcription factor (MiT) family aberration-associated renal cell carcinoma (MiTF-RCC), a renal tumor subtype known to affect children as well as adult patients. We developed a TRIM63 biomarker assay using a novel single-molecule chromogenic RNA in situ hybridization (RNA-ISH) technology. High expression of TRIM63 was observed in all classes of MiTF-RCC, including RCC with translocations involving TFE3 gene located on chromosome Xp11.2, RCC with translocations involving TFEB gene on chromosome 6p21, and RCC with TFEB gene amplification. We showed that by applying TRIM63 biomarker assay, potential diagnostic pitfalls of TFE3/TFEB FISH assay can be addressed. For example, TRIM63 positive/FISH negative cases were seen to carry cryptic intrachromosomal Xp11.2 inversion resulting in RBM10-TFE3 gene fusion; another TRIM63 positive/FISH negative case was found to have low copy number gain below the clinical threshold for RCC with TFEB amplification. By evaluating the TRIM63 expression in renal tumor cases that are clinically and morphologically suspicious for MiTF-RCC, we also discovered a novel gene fusion variant involving TFE3 and ZC3H4 genes in a RCC case with high TRIM63 expression. The performance of TRIM63 biomarker RNA-ISH assay is highly concordant to the TFE3/TFEBbreak-apart FISH assay, the current clinical and gold standard for MiTF-RCC diagnosis. TRIM63 staining was also found to be superior to the clinical immunohistochemistry (IHC) markers currently employed for a diagnostic workup of MiTF-RCC, including Melan-A, HMB-45, and Cathepsin K.
E3 ubiquitin ligases are known to be involved in oncogenic transformation. The TRIM family genes are known to play an important role in autophagy that contributes to cancer. The potential oncogenic role of TRIM63 ubiquitin ligase activity in MiTF-RCC and transcriptional regulation of TRIM63 by MiTF transcription factor are exciting new avenues that need further exploration.
The recent advances in RNA-ISH technology have enabled our group to utilize this tool for rapid biomarker development process. In our experience, this method, through evaluation of signals under the microscope, provides simultaneous qualitative and quantitative estimations of gene expression (i.e., the target RNA of interest). In addition to the visualization of biomarker expression, its distribution within the whole tumor tissue area, and a comparison to background benign tissue, can be faithfully performed Consistent assay performance, comparable results to FISH methodology and user-friendly workflow makes RNA-ISH very appropriate in a research setting, with great potential to be incorporated into the clinical pathology laboratory testing systems.
In summary, TRIM63 is a sensitive and specific biomarker for MiTF-RCC. TRIM63 RNA-ISH could serve as a clinical ancillary tool for the diagnostic workup of MiTF-RCC, and a potential screening tool for the purposes of clinical trials enrolling patients with MiTF-RCC.
Written by: Xiao-Ming Wang, PhD, Rahul Mannan, MBBS, MD, Saravana M. Dhanasekaran, PhD, Rohit Mehra, MD, Department of Pathology, University of Michigan Medical School, Ann Arbor, MI, USA
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