The p53 gene has been demonstrated consistently to be significantly altered in gastric adenocarcinomas. Allelic loss occurs in over 60 percent of patients, and mutations are identified in approximately 30 to 50 percent of patients depending on the mutational screening method employed [i.e., single-stranded conformational polymorphism (SSCP) assay or degenerative gradient gel electrophoresis assay] and sample sizes.56 Some mutations of p53 have even been identified in early dysplastic and apparent intestinal metaplasia gastric lesions. In general, however, alterations of this gene occur more frequently in the advanced stages of dysplasia in both histopathologic subtypes. The spectrum of mutations in this gene within gastric tumors appears similar to that which occurs in other cancers with a predominance of base transitions, especially at CpG dinucleotides. Inactivation of this important cell cycle regulator appears to confer a growth advantage and allow clonal expansion of transformed cells. Many studies have used immunohistochemical analysis of tumors in an effort to detect excessive expression of p53 as an indirect means to identify mutations of this gene, but this assay does not appear to have consistent prognostic value in patients with gastric cancers.57,58
Microsatellite instability has been found in a significant portion of sporadic gastric carcinomas.59-61 Variability in classification of instability or histopathologic subtype and number of loci examined in studies account for some variation of this phenotype's frequency, with a trend toward more frequent occurrence in intestinal-type cancers at more advanced stages observed, although noted in early lesions as well (i.e., adenomas). The degree of genome-wide instability also varies, with more severe instability (e.g., >2 abnormal loci) associated with subcardial intestinal or atypical types. A negative association with p53 alterations also has been suggested, indicating different paths of alterations accumulating in individual gastric tumors. Studies have indicated less frequent lymph node or vessel invasion, prominant lymphoid infiltration, and better prognosis in those gastric cancers which displayed significant microsatellite instability.59,62,63 However, it remains to be proven if this phenotype is a prognostic marker for improved survival, as suggested in colon cancers. The alterations responsible for producing this phenotype in a subset of sporadic gastric cancers remain to be elucidated.
At least one important target of the instability in those cancers displaying abnormally sized microsatellites appears to be the transforming growth factor beta (TGF-β) type II receptor. A study of gastric cancers displaying the microsatellite instability phenotype revealed that a majority (5 of 7) contained mutated TGF-β type II receptors at a polyadenine tract within its gene.64 Moreover, altered TGF-β type II receptor genes could be found in gastric cancers not displaying microsatellite instability. Several gastric cancer cell lines resistant to the growth inhibitory and apoptotic effects of TGF-β were shown to have altered TGF-β type II genes (deletions and amplifications) and transcripts (truncated or absent).65 Thus TGF-β type II receptor mutation appears to be a critical event in the development of at least a subset of gastric cancers, allowing escape from the growth control of TGF-β. Additional genes with simple tandom repeat sequences within their coding regions found to be altered in gastric cancers displaying microsatellite instability include BAX, IGFRII, hMSH3, hMSH6, and E2F-4. 66-68
As mentioned earlier, E-cadherin germ-line mutations have been found in several large kindreds exhibiting a strong predisposition to diffuse gastric cancer development. Several sporadic gastric cancers also have displayed altered E-cadherin, mainly in diffuse cases. Reduced E-cadherin expression determined by immunohistochemical analysis was noted often (92 percent of 60 patients) in gastric carcinomas and observed to be significantly associated with diffuse-type cancers and more undifferentiated neoplastic cells (i.e., signet ring cells).69 Genetic abnormalities of the E-cadherin gene (located on chromosome 16q22.1) and transcripts also have been demonstrated in diffuse gastric cancers.70 Half of 26 diffuse gastric carcinomas had abnormal E-cadherin transcripts detected by reverse transcription PCR (RT-PCR) analysis that were not seen in noncancerous tissue from the same patients. Moreover, a study of 10 gastric cancer cell lines displaying loose intercellular adhesion found absent E-cadherin transcripts in four lines and insertions or deletions in two other lines.71 Splice-site alterations producing exon deletion and skipping, large deletions including allelic loss, and point mutations of the E-cadherin gene were all demonstrated in these diffuse-type cancers, some even exhibiting alterations in both alleles. In comparison of RT-PCR products from normal tissue and tumor tissue from patients, allelic expression imbalance of E-cadherin also has been shown in a porportion (42 percent of 35 informative cases) of gastric carcinomas.72 E-cadherin is a transmembrane, calcium-ion-dependent adhesion molecule (important in epithelial cell homotypic interactions) that, when decreased in expression, is associated with invasive properties.73 Additionally, α-catenin, which binds to the intracellular domain of E-cadherin and links it to actin-based cytoskeletal elements, was noted to have reduced immunohistochemical expression in 70 percent of 60 gastric carcinomas and correlated with infiltrated growth and poor differentiation.74
LOH studies suggest that chromosome 5q harbors at least one tumor-suppressor gene important in the development of gastric cancers.49,51,52,75,76 The exact target(s), however, of this loss in gastric tumors is not fully clarified. Several somatic APC mutations, mostly missense in nature and of relatively low frequency, have been reported in Japanese patients with gastric adenocarcinomas and adenomas using ribonuclease protection or SSCP assays for partial screening.77 On the other hand, several other reports including Japanese patients have not identified significant APC mutations in gastric carcinomas on similar partial screening analysis of the commonly mutated region and include direct nucleotide sequencing and the sensitive in vitro synthesis protein assay.76,78-80 Interestingly, an increased risk of gastric cancer associated with familial adenomatous polyposis (patients with germ-line APC mutations) has been reported in high-risk regions such as Asia, 81,82 whereas no increased risk was exhibited in other populations.83,84 Significant allelic loss (30 percent) at the APC locus suggests the existence of a tumor-suppressor gene important in gastric tumorigenesis nearby. Indeed, alternative loci have been mapped to commonly deleted regions in gastric cancers (the interferon regulatory factor 1 loci and D5S428)75 and esophageal cancers (5q31.1).85 Thus future studies should help define the important gene(s) on chromosome 5q, which is critically involved in gastric tumorigenesis.
The targets of loss on other chromosomes implicated to harbor important tumor-suppressor gene(s) in gastric cancer also remain to be defined. Significant allelic loss (60 percent) has been noted at the DCC locus on chromosome 18q in gastric cancers.50 Only one of 35 gastric cancers contained an intragenic mutation of Smad4 along with allelic loss, suggesting that this MADD homologue gene is infrequently altered in gastric tumorigenesis.86
Evidence of a tumor-suppressor loci on chromosome 3p has accumulated from a variety of studies, including allelic loss in primary gastric tumors (46 percent) and homozygous deletion in a gastric cancer cell line (KATO III).87 A candidate tumor-suppressor gene, FHIT, recently isolated from the FRA3B site at 3p14.2, was reported to have abnormal transcripts of deleted exons in 5 of 9 gastric cancers in addition to transcript abnormalities noted in esophagus, colon, lung, and head and neck cancers as well.88-90 One somatic missense mutation was identified in exon 6 of the FHIT gene during a coding region analysis of 40 gastric carcinomas.91 Significant abnormalities of the FHIT gene were not observed in a study of 31 colorectal cancer patients.92 Addtional studies should help clarify the role FHIT plays in gastric tumorigenesis.
The c-met gene encodes a tyrosine kinase receptor for the hepatocyte growth factor. Amplification of the c-met gene was reported to be associated with scirrhous-type gastric cancers.93 Northern blot analyses of gastric cancer cell lines and resected primary carcinomas compared with paired nonneoplastic tissue showed overexpression of a 7.0-kb transcript of the c-met gene in 48 percent of 31 cancers, predominantly of the well-differentiated type.94 Moreover, a 6.0-kb c-met transcript appeared to be preferentially expressed in scirrhous gastric tumor cells and correlated with latter stages of tumor development. Tumor and stromal cell interactions have been implicated with this growth factor and receptor signal system as well as involvement of multiple others, including epidermal growth factor (which is expressed in approximately one-quarter of gastric cancers), transforming growth factor alpha (TGF-α), interleukin-1a, criptor, amphiregulin, platelet-derived growth factor, K-sam, and others.53,95 Telomerase activity has been detected by a PCR-based assay frequently in the late stages of gastric tumors (85 percent of 66 patients) and is associated with a poor prognosis.96 The expression of telomerase, a ribonucleoprotein DNA polymerase, and stabilization of telomeres have been noted to be concomitant with immortalization in tumor cells.97 Specific alterations and the true prevalence of significant changes in these genes or gene products in gastric tumors remain to be characterized.
Another potential marker of poor prognosis is overexpression of c-erbB-2, a transmembrane tyrosine kinase receptor proto-oncogene. Amplification of c-erbB-2 has been demonstrated in a small subset of gastric cancers, approximately 10 percent.98 Several reports have shown amplification or increased expression of erbB-2 immunohistochemically in gastric tumors to be associated with a worse prognosis.99 Furthermore, enhanced expression of erbB-2 recently has been demonstrated to occur more frequently in gastric cancers displaying microsatellite instability.100 The specific genetic or epigenetic alterations underlying these immunohistochemical findings remain to be characterized.
A number of other alterations have been reported in gastric carcinomas that remain to be defined, as well as the role they play in gastric tumorigenesis. Several splice variants of a transmembrane glycoprotein, CD44, seem to be preferentially expressed in gastric tumors cells.101 Membrane-type matrix metalloproteinase was expressed preferentially in some gastric cancer cells with colocalization and activation of the zymogen proMMP-2.102 Both loss and overexpression of Bc1-2 and nm23 have been reported in several gastric cancers, making their role unclear. Amplification of cyclin E and increased plasminogen activation have been reported as well in several gastric tumors.103 A somatic mitochondrial deletion of 50 bp was even demonstrated in four gastric adenocarcinomas.104
Activation of the oncogene K-ras appears to be rare in gastric tumorigenesis.79,105,106 Although allelic loss was noted in 18 percent of gastric tumors at the locus for p16 on chromosome 9p, no inactivating somatic mutations were detected in over 70 patients screened by PCR-SSCP analysis.107 No methylation abnormalities or genetic alterations of cycle regulators such as p16, p21, or p27 have as yet been reported.