Detection of metastatic head and neck squamous cell carcinoma using the relative expression of tissue-specific mir-205

Abstract

The presence of cervical lymph node metastases in head and neck squamous cell carcinoma (HNSCC) is the strongest determinant of patient prognosis. Owing to the impact of nodal metastases on patient survival, a system for sensitive and accurate detection is required. Clinical staging of lymph nodes is far less accurate than pathological staging. Pathological staging also suffers limitations because it fails to detect micrometastasis in a subset of nodal specimens. To improve the sensitivity of existing means of diagnosing metastatic disease, many advocate the use of molecular markers specific for HNSCC cells. MicroRNA (miRNA) are short noncoding segments of RNA that posttranscriptionally regulate gene expression. Approximately one third of all miRNA will exhibit substantial tissue specificity. Using a quantitative reverse transcription-polymerase chain reaction-based assay, we examined the expression of microRNA-205 (mir-205) across tissues and demonstrated that its expression is highly specific for squamous epithelium. We applied this assay to tissue samples, and we could detect metastatic HNSCC in each positive lymph node specimen, whereas benign specimens did not express this marker. When compared to metastases from other primary tumors, HNSCC-positive lymph nodes were distinguishable by the high expression of this marker. Using an in vitro lymphoid tissue model, we were able to detect as little as one squamous cell in a background of 1 million lymphocytes. By combining the sensitivity of quantitative reverse transcription-polymerase chain reaction with the specificity of mir-205 for squamous epithelium, we demonstrate a novel molecular marker for the detection of metastatic HNSCC.

Figure 1

Tissue-specific expression of mir-205. The relative fold expression difference of mir-205 in each sample relative to the calibrator sample (spleen).

Figure 2

Expression of mir-205 in cell lines and tissue samples. (A) The expression of mir-205 in the HNSCC cell lines after normalization to the NHOK cell line. (B) The expression of mir-205 in primary tissue from various anatomical subsites after normalization to normal mucosal tissue.

Figure 3

Expression of mir-205 in metastatic lymph node samples. The fold expression difference in mir-205 for each specimen relative to the benign node samples (error bars indicate SD for each sample).

Figure 4

Sensitivity of qRT-PCR to detect mir-205. The relative expression of each cell dilution when compared to the Jurkat-only sample.