Comparative Gene Expression and miRNA Expression Patterns in Chromophobe Renal Cell Carcinoma (ChRCC) and Oncocytoma (OC)
Milon Amin, Anil V Parwani, Somak Roy, Sheldon Bastacky, William LaFramboise, Rajiv Dhir. University of Pittsburgh Medical Center, Pittsburgh, PA
Background: ChRCC and OC are distinct neoplasms arising from the intercalated duct of collecting tubules. Significant differences in clinical outcome warrant a definitive diagnosis that may be challenging due to overlapping morphologic and immunophenotypic features. We compared mRNA and microRNA transcriptional profiles of both tumors using high density expression arrays.
Design: Total RNA was purified from microdissected specimens using the Qiagen miRNeasy kit with spectrophotometric absorption: 260/280 >1.8 and RIN value >8.0. RNA (1ug) was labeled with Hy3 for hybridization on miRCURY LNA arrays (Exiqon, Woburn, MA) for 18 hours followed by stringent wash and scan with probe readout classification against the Sanger miRBase (v.11.0). Message RNA (500ng) underwent cDNA synthesis and in vitro transcription using Ambion WT Expression assays (Ambion Inc, Austin, TX) followed by fragmentation and hybridization on Human Exon 1.0 ST arrays (Affymetrix Corp.,Santa Clara, CA) for 18 hours. Arrays were washed, stained and scanned (Affymetrix Fluidics Station 450, Scanner 3000) after hybridization. Signal intensity was calculated by Microarray Suite (v.5.0). Statistical comparisons (ANOVA) were performed (Partek Genomics Suite, St. Louis, MO) with false discovery rate:q value=0.1, -2.0>fold-change>2.0 and removal of comparisons within 5% of background signal intensity.
Results: 856 and 38 mRNA transcripts had significantly altered expression in ChRCC and oncocytoma, respectively. ChRCC exclusively harbored altered expression of 785 transcripts. Conversely, there were no statistically significant alterations in mRNA expression unique to OC. Further analysis revealed 60 tumor suppressor genes and 30 oncogenes with significantly altered expression in ChRCC. Preliminary pathway analysis segregated the above genes into the following signaling pathways: MAP Kinase, Insulin, Chemokine, Wnt, Hedgehog, Notch, VEGF, PPAR, ERBB and TGF-beta. miRNA expression profiling revealed 153 statistically significant changes specific to ChRCC with no changes in OC expression. Analysis of common miRNAs altered in both tumor types compared to normal tissue demonstrated that miRNA200c and miRNA141 were overexpressed in ChRCC and underexpressed in OC.
Conclusions: Assessment of differential mRNA and miRNA expression in ChRCC and OC provides novel markers for discrimination of both tumors. Absence of statistically significant alterations in mRNA expression in OC supports its accepted benign nature. Identifying pathways with aberrant activity in ChRCC has implications for targeted therapy.
Category: Genitourinary (including renal tumors)
Wednesday, March 6, 2013 9:30 AM
Poster Session V # 140, Wednesday Morning