Identification of distinct regions of allelic loss on chromosome 13q in nasopharyngeal cancer from paraffin embedded tissues

Abstract

Our main purpose was to identify tumor suppressor gene loci on chromosome 13 responsible for nasopharyngeal cancer (NPC) development by analyzing loss of heterozygosity (LOH) and RB protein expression in paraffin embedded tissues. Normal and tumor DNA were extracted from microdissected samples, and their whole genomes were amplified using degenerate oligonucleotide primers. The polymerase chain reaction (PCR) products were analyzed by repeated amplification using primers derived from 16 microsatellite regions spanning the long arm of this chromosome. Among 50 informative cases, LOH was observed in 44 tumors. Thirty-one tumors displayed partial loss and provided an informative basis for detailed deletion mapping. Three minimal regions of loss were delineated; the first flanked by D13S120 and D13S219, the second by D13S126 and D13S119, and the third by D13S137 and 13qter. These 3 regions were linked to BRCA2 on 13q12, RB1 on 13q14, and 13q14.3-ter, respectively. Seven and 4 cases showed LOH either on 13q12 or 13q14, respectively. Nineteen cases showed LOH of both loci separately. One NPC displayed 13q12 and 13q14.3-ter LOH. RB protein expression was detectable in 76% of the cases. Ten out of 15 cases with the allelic losses limited to 13q14 showed RB protein expression. Contrasting that, 6 out of 7 cases devoid of RB protein expressions showed 13q14LOH. In conclusion, 13qLOH, involving 3 tumor suppressor gene loci, appears to be a frequent genetic event occurring during NPC development. However, other tumor suppressor genes besides RB1, may be responsible for the majority of 13q14LOH. Int. J. Cancer 83:210-214, 1999. Wiley-Liss, Inc.Nasopharyngeal cancer (NPC) is one of the most common cancers in Asia, especially in Southern China and Southeast Asia (Fandi et al., 1994;Voravud, 1990). The development of this type of cancer appears to be caused by Epstein-Barr virus (EBV) infection with subsequent multiple genetic alterations in the nasopharyngeal epithelium (Liebowitz, 1994;Mutirangura et al., 1997). Frequently, loss of heterozygosity (LOH) has been observed on chromosomes 3, 9, 11, 13 and 14, suggesting a potential location for essential tumor suppressor genes (Mutirangura et al., 1997). Several previous studies have defined the locations of the allelic losses on chromosomes 3, 9, 11 and 14 Huang et al., 1994;Hui et al., 1996;Mutirangura et al., 1998). The main objective of our study has been to analyze LOH loci on chromosome 13 in NPC tissues. Since the majority of NPC tissues exhibit either massive lymphocyte infiltration or contamination with normal tissue, we performed the microsatellite analysis from microdissected paraffin embedded NPC tissues.LOH on chromosome 13q has frequently been observed in primary cancers of the lung (Tamura et al., 1997), prostate (Cooney et al., 1996;Li et al., 1998), skin (Sikkink et al., 1997, breast (Hamann et al., 1996), head and neck (Maestro et al., 1996;Nawroz-Danish et al., 1998), oral cavity (Ogawara et al., 1998) and liver (Kuroki et al., 1995). This may be due to the loc...

Gene expression profiling of nasopharyngeal carcinoma reveals the abnormally regulated Wnt signaling pathway

“…Previous reports showed that environmental factors such as the intake of salted food, Epstein-Barr Virus (EBV) infection, and the function of genetic factors were thought to be important in the etiology of NPC [3]. Loss of heterozygosity (LOH) studies unveiled there are frequent allelic losses on chromosomes 3p, 9p, 11q, 13q, and 14q in NPC [4][5][6][7][8][9]. Chromosome regions 4p15.1-q12 and 3p21 have also been identified as functional NPC susceptibility loci by linkage analysis of Chinese pedigrees [10][11][12].…”

Distinctive pattern of LINE-1 methylation level in normal tissues and the association with carcinogenesis

“…We used 5 0 UTR of LINE-1.2 sequence from NCBI Accession Number M80343. DNA isolation and extraction from leukocytes, sera, and unstained microdissected paraffin-embedded tissues and treatment with bisulfite have been described previously (Herman et al, 1996;Xiong and Laird, 1997;Mutirangura et al, 1998Mutirangura et al, , 1999. Bisulfited DNA was subjected to 35 cycles of PCR with two primers, 5 0 -CCGTAAGGGGTTAGGGAGTTTTT-3 0 and 5 0 -RTAAAACCCTCCRAACCAAATATAAA-3 0 , with an annealing temperature of 501C.…”

Functional Approaches to Localize and Identify Tumor Suppressive Regions Required for Development of Nasopharyngeal Carcinoma

“…While the identification and isolation of specific TSGs in NPC development is still forthcoming, molecular studies utilizing microsatellite typing and loss of heterozygosity (LOH) approaches implicate the involvement of TSGs residing on chromosomes 3,9,11,13,14, and 16 in NPC. [28][29][30][31][32][33][34][35][36][37][38][39] High frequencies and consistent occurrence of LOH on chromosomes 3, 9, 13, and 14, in particular, highlight the importance of the candidate TSGs mapping to these chromosomes in NPC. Conventional cytogenetics and current comparative genomic hybridization (CGH) data support these molecular findings concerning involvement of numerous regions of multiple chromosomes in NPC.…”