microRNA Expression in Archived FFPE Head and Neck Squamous Cell Carcinomas Utilizing Multiplex miRNA Expression Assays
Laura J Tafe, Thomas H Davis, Mary C Schwab, Gregory J Tsongalis. Dartmouth Hitchcock Medical Center, Lebanon, NH
Background: Micro RNAs (miRNA) play a role in regulating the expression of genes that are important in cellular differentiation, proliferation, survival and cell death. Multiple studies have established that many cancer types, including leukemias and solid tumors, exhibit distinct miRNA expression profiles that likely play a role in tumorigenesis. Limited studies have examined miRNA expression in head and neck squamous cell carcinomas (HNSCC). The aims of this study were twofold: first, to determine if FFPE is an acceptable sample type for the multiplex miRNA expression assays, and, second, to identify miRNA expression patterns that may be characteristic of HNSCC when compared to matched normal tissues.
Design: Total RNA was extracted and purified from 24 archived FFPE matched tumor and normal HNSCC (6 laryngeal, 6 oral tongue) using the Qiagen RNeasy FFPE kit. Approximately 100ng of total RNA was run in a final reaction volume of 30ul using nanoString's nCounter miRNA Expression Assay Kit on the nCounter platform. This assay detects over 700 miRNAs in a multiplex format. The geometric mean of the top 150 expressing miRNAs was taken for normalization. The expression of miRNAs in tumor tissues was compared to the matched normal counterparts.
Results: Two of 24 samples (1 tumor, 1 normal) showed low counts, indicating possible degradation, therefore, a total of 10 sample pairs had analyzable results (4 laryngeal and 6 tongue). The tumors expressed a median of 147 miRNAs (range 82-435) and the normal tissues expressed a median of 133 miRNAs (range 107-224). Several patterns of expression were observed: miR-21 was upregulated in 7/10 cases (3-14 fold) including 5/6 tongue tumors. MiR- 205 and miR-1274b were upregulated in 5/10 total cases (3-10 fold) and in 4/6 tongue tumors (3-4 fold), respectively. MiR-451and miR-125b showed downregulation in 6/10 (3-72 fold) and 5/10 (3-12 fold) total tumors, respectively.
Conclusions: In this study, we have shown that the nCounter miRNA Expression Assay Kit can be successfully run on HNSCC FFPE archival tissue. Our findings of the upregulation of miR-21 in HNSCC are consistent with prior reports in the literature. In addition, we identified other miRNAs that may play a regulatory role in the tumorogenesis of HNSCC and that may have a tumor site specific profile (i.e. tongue vs. larynx) that lend themselves to further investigation.
Category: Head & Neck
Wednesday, March 21, 2012 9:30 AM
Poster Session V # 184, Wednesday Morning