Allelic Expression Imbalance of TP53 Mutated and Polymorphic Alleles in Head and Neck Tumors Federica Ganci, 1, * Salvatore Conti, 1, * Giulia Fontemaggi, 1 Valentina Manciocco, 2 Sara Donzelli, 1 Renato Covello, 3 Paola Muti, 5 Sabrina Strano, 4 Giovanni Blandino, 1 and Giuseppe Spriano 2 Abstract TP53 is the most widely mutated gene across all cancer types. In head and neck cancer, approximately half of the tumors are found to contain TP53 mutations, which are correlated to an increased risk for locoregional recur- rence and poor outcomes. In this study a mutational profiling of TP53 exons 5–8 was performed on tumor, peritumor and normal tissues from 57 HNSCC patients by direct sequencing of genomic DNA and cDNA. Cloning/sequencing in tumors carrying multiple TP53 mutations and semiquantitative SNaPShot mutation assay was performed in order to assess eventual allelic expression imbalances (AEI). We identified 24 out of 57 HNSCC patients (42%) carrying TP53 mutations and 5 patients carrying the R213R polymorphism. Cloning of the genomic DNA encompassing TP53 exons 5–8 from tumors with multiple TP53 mutations revealed that alleles carrying different types of TP53 mutations are present in these tumors. TP53 missense and nonsense mutations exhibit higher and lower TP53 transcript abundance compared to wild-type TP53 allele, respectively. Interestingly, three out of four patients with the R213R polymorphism analyzed were found positive for TP53 loss of heterozygosity (LOH) and also presented higher transcript abundance than the wild-type counterpart, specifically, in the tumor tissue and not in peritumor or normal tissues. HNSCC tumors present heterogenic cell populations carrying different TP53 mutations. All HNSCC samples analyzed show an alteration in the ex- pression of mutated TP53 mRNA compared to the wild-type allele, most likely independently from the TP53 hemizygous status. The higher expression of R213R TP53 polymorphic allele in cancer tissue compared to normal tissue demonstrates a noninherited variation in allelic expression, independently from its mutation status for exons 5–8, suggesting a potential contribution to TP53 expression in HNSCC disease. Introduction H ead and neck squamous cell carcinomas (HNSCCs) are a heterogeneous group of tumors that arise from the epithelium of the upper aerodigestive tract. HNSCC is the eighth most common human cancers worldwide and is associated with high alcohol and tobacco use (Pai and Westra, 2009). Infection with human papillomavirus high- risk (HPV) types is also related to the development of HNSCC, independently from tobacco and alcohol use. HPV infection has demonstrated to play a role in the molecular pathways through its viral oncoproteins, E6 and E7. In par- ticular, E6 increases degradation of the p53 protein (Smith et al., 2010). The p53 tumor suppressor gene (TP53), is the most frequent target in genetic alterations in human cancers, with a preva- lence of 20–60% in HNSCC (Edstro ¨ m et al., 2001; Nogueira et al., 1998). The region containing exons 5–8, encoding for the evolutionary conserved DNA-binding domain, represents the major site of TP53 mutations. After mutation in one TP53 allele, the remaining wild-type allele is often deleted and therefore, the mutant phenotype is expressed (Kiuru et al., 1997; Yin et al., 1993). Countless evidences has demonstrated that at least cer- tain mutant forms of p53 protein may possess gain of function activity, thereby positively contributing to cancer progression. When the mutational analysis of TP53 and clinical outcomes in HNSCC were correlated, the results obtained were contradic- tory. A number of studies reported that TP53 gene mutations 1 Translational Oncogenomics Unit, Regina Elena Cancer Institute, Rome, Italy. 2 Otolaryngology Department, Regina Elena Cancer Institute, Rome, Italy. 3 Pathology Department, Regina Elena Cancer Institute, Rome, Italy. 4 Molecular Chemoprevention Group, Scientific Direction, Regina Elena Cancer Institute, Rome, Italy. 5 Scientific Direction, Regina Elena Cancer Institute, Rome, Italy. *These authors contributed equally to this work. OMICS A Journal of Integrative Biology Volume 15, Number 6, 2011 ª Mary Ann Liebert, Inc. DOI: 10.1089/omi.2010.0142 375