Identification of genes overexpressed in head and neck squamous cell carcinoma using a combination of complementary DNA subtraction and microarray analysis

Objectives/Hypothesis: To discover unique genes specific for squamous cell carcinoma of the head and neck for eventual development as tumor markers an d vaccine candidates. Study Design: Molecular biological analysis of fresh- frozen head and neck squamous cell cancer (HNSCC), Methods: A subtractive L ibrary was made from two HNSCC and six normal tissues using a polymerase ch ain reaction (PCR)-based approach. Genes from this library were PCR amplifi ed and placed on a microarray glass slide. RNA was prepared or obtained fro m 16 fresh-frozen HNSCC and 22 normal tissue sources. Fluorescent probes we re made from the polyA+ RNA derived from the tumor and normal tissues. The probes were hybridized to the glass slides and excited by a tuneable laser One hundred seven of the genes showing the highest differential fluorescenc e value between tumor and normal tissue were identified by sequence analysi s. Results: Thirteen independent genes were found to be overexpressed in tu mor tissues, Of these, nine were previously known: keratins K6 and K16, lam inin-5, plakophilin-1, matrix metalloproteinase-2 (MMP), vascular endotheli al growth factor, connexin 26, 14-3-3 sigma, and CaN19, The level of polyA RNA of these genes in the tumors was significantly different from the leve ls in normal tissue (P <.05). Four previously unidentified genes were also discovered to have increased expression in tumor tissue. Comparing the tota l tumor group (n = 16) to the normal group (n = 22), only one of these gene s showed significant overexpression. Conclusion: We report the identificati on of nine known genes that are significantly overexpressed in HNSCC as com pared to normal tissue using subtractive and microarray technology, In addi tion, we present four previously unidentified genes that are overexpressed in a subset of tumors. These genes will be developed as tumor markers and v accine candidates.