Loss of heterozygosity (LOH) in a cell represents the loss of one parent’s contribution to part of the cell’s genome. Normally, there are slight differences between the two copies of a gene, since one comes from each parent. When this heterozygosity is lost, it signifies that a muta tion likely exists. The LOH can arise via several pathways, including deletion, gene conversion and chromosome loss. Loss of heterozygosity of polymorphic loci in a chromosomal fragment implies the presence of putative tumor suppressor genes. Loss of heterozygosity can be identified in cancers by noting the presence of heterozygosity at a genetic locus in an organism’s germline DNA, and the absence of heterozygosity at that locus in the cancer cells. This is often done using polymorphic markers, such as microsatellites or single nucleotide polymorphisms for which the two parents contributed different alleles. A genomewide scan for LOH in a diverse collection of HCC samples from Europe and Asia has shown the highest per centages in loci at chromosomes 8p23, 4q22-24, 4q35, 17p13, 16q23-24, 6q27, 1p36 and 9p12-14 [30]. The most frequently affected chromosome arm is 1q, with rates of amplification ranging between 58 and 78% in HCC [31]. Other chromosome arms commonly altered with gains include 6p, 8q and 17q, and allelic losses are found at 1p, 4q, 6q, 8p, 9p, 13q, 16q and 17p [32].

Tumor-Suppressor Genes. Tumor-suppressor genes normally function to inhibit cellular proliferation. They are considered to be ‘recessive,’ since loss of function of both alleles is necessary to generate the mutant phenotype. A significant percentage of HCC patients carry mutations in the TP53 tumor-suppres-sor gene encoding the p53 protein [33]. In HCC, the rate of mutations of p53 ranges from 0% (in 129 HCC samples from Spain) to 67% (in 15 HCC samples from Senegal). There is a remarkable differential mutation rate according to geographic area: higher rates of mutation were documented in West Africa and Southeast Asia and lower rates in western countries. The rate of mutations of p53 in 107 geographically and ethnically diverse HCC samples was 25 and 12% in high- and low-aflatoxin exposure regions, respectively [31]. In most cases, loss of p53 function occurs through allelic deletions at chromosome 17p13 or due to missense mutations within the specific DNA-binding domain. The R249S mutation (G/C>T/A transversion) in p53 is frequent in some regions with high aflatoxin exposure [30]. Although the p53 gene promoter does not contain a CpG island, an increase in promoter methylation of the p53 that led to reduced gene expression in human HCC has been described [34].

Mutations have been found in the gene encoding the axis inhibition protein 1 (AXIN1) gene (16p13.3) in 7-10% of patients with HCC which led to aberrant activation of Wnt signaling [33]. The canonical Wnt pathway describes a cascade that involves translocation of â-cate nin from the cell membrane into the nucleus where it regu lates specific target genes, including c-myc and cyclin D. The AXIN protein is part of a protein complex involved in the degradation ofâ-catenin [31]. Mutations in the AXIN1 in the absence of mutation in the catenin (cadherin-associ ated protein) â1 (CTNNB1) gene (codes for â-catenin) could prevent degradation and lead to nuclear accumula tion of â-catenin. In addition, over expression of wildtype AXIN1 can sequester both normal and mutated â-catenin in the cytoplasm, which leads to apoptosis [30].

Oncogenes: the â-Catenin Pathway. The most fre quently mutated oncogene in human HCC is CTNNB1 (located at 3p21) encoding â-catenin protein. The â-cate nin is involved in cell-cell adhesion by association with E-cadherin and also in transmission of the proliferative/ survival signal during embryonic development [30]. In the absence of Wnt signaling, â-catenin is phosphorylated by functional interactions with glycogen synthase kinase (GSK)-3â, AXIN, conductin (AXIN2) and the APC pro tein, leading to its degradation by the ubiquitin-proteasome system [35]. The activation of the Wnt signal induces â-catenin stabilization through inhibition of GSK-3â activ ity. After translocation to the nucleus, â-catenin is able to activate a number of genes including c-myc, cyclin D1, WNT1 inducible signaling pathway protein 2 (WISP), fibronectin and matrix metalloproteinase genes. The CTNNB1 mutations in human HCC include mostly mis sense mutations as well as interstitial deletions of the CTNNB1 exon 3 with a prevalence of 18-41% [30]. Another gene possibly involved in hapatocarcinogenesis is Frizzled (FZD) which codes for a transmembrane recep tor in the Wnt-signaling cascade. The FZD signals to â-catenin to escape its association with E-cadherin, thus influencing cell-cell adhesion [31].