ABSTRACT:

Gastric adenocarcinoma is a leading cause of cancer mortality world- wide. Yet, the underlying molecular events important in the development of this cancer are largely undefined. Thus, we performed a comprehensive survey for allelic loss on our panel of xenografted human gastric carcinomas. Contaminating normal stromal cells of primary cancers often limit mutational analyses. Xenografted samples of our gastric carcinomas provided optimally enriched tumors for neoplasia that clearly and sensitively demonstrated genetic alterations. Additionally, total absence of allelic signals in these xenografted samples confirmed true loss of alleles rather than just allelic imbalance. Analysis of at least two highly polymorphic microsatellite markers per nonacrocentric chromosomal arm in our xenografted human gastric carcinomas demonstrated significant loss of heterozygosity well above background levels at 3p, 4p, 5q, 8p, 9p, 13q, 17p, and 18q. Several of these loci represent novel findings of significant loss in gastric cancers. On chromosome 17p, p53 is known to be inactivated either by mutation or deletion in a majority of gastric carcinomas. The critical target(s) of inactivation in gastric cancers at these other loci remain to be characterized.

Figure. Allelotype of xenografted gastric cancers.

  ABSTRACT:

The genetic alterations underlying the development of gastric and gastro-esophageal carcinoma remain largely undefined. DNA copy number changes were determined by comparative genomic hybridization in eight xenografts of proximal gastric and gastro-esophageal junction adenocarcinomas of the intestinal type. All tumors exhibited DNA copy number changes, with a total of 139 changes detected (range, 11-24 per tumor; mean = 17), indicating numerous and widespread alterations within these cancers. Gains (65%) in DNA copy number were more frequent than losses (35%). Our most striking finding was gain (all eight cases) or high-level amplification (four cases) in 20q, with a minimal common overlapping region at 20q13. Other frequent gains were observed at 6p, 7q, and 17q (six cases each) and at 1q, 2q, and 8q (five cases each). Frequent losses were observed at 4q and 5q (six cases each) and at 9p (five cases). No differences in DNA copy number changes were seen in tumors arising from the gastro-esophageal junction compared to those of the proximal stomach. The presence of common and consistent DNA copy number changes in these tumors implicate a number of chromosomal regions that may harbor important genes that are involved in tumorigenesis of the proximal stomach and gastro-esophageal junction.

Figure. Summary of DNA copy number gains and losses....

 ABSTRACT:

Microsatellite instability (MSI) is observed in 13-44% of gastric carcinoma. The etiology of MSI in gastric carcinoma has not been clearly defined. To assess the role of mismatch repair in the development of MSI in gastric cancer, expression of hMSH2 and hMLH1 was explored. We examined 117 gastric carcinomas for MSI and observed instability at one or more loci in 19 (16%) of these tumors. Of the 19 tumors with MSI, nine exhibited low-rate MSI (MSI-L) with instability at 33% of loci examined. Immunohistochemical staining for hMLH1 and hMSH2 was performed on eight of the tumors with MSI-H, five with MSI-L, and 15 tumors without MSI. All eight tumors with MSI-H showed loss of staining for either hMLH1 (n = 5) or hMSH2 (n = 3). In contrast, tumors with MSI-L or without MSI all showed normal hMSH2 and hMLH1 protein expression patterns. Moreover, all eight of the tumors with MSI-H also showed instability at BAT-26, whereas none of the MSI-L tumors or tumors without instability showed instability at BAT-26. These findings suggest that the majority of high-level MSI in gastric cancer is associated with defects of the mismatch repair pathway. Although larger studies are needed, BAT-26 appears to be a sensitive and specific marker for the MSI-H phenotype in gastric carcinoma.

Figure. Representative examples of loss of hMSH1 expression in two gastric carcinomas.

  ABSTRACT:

Human gastric carcinoma shows a higher prevalence of microsatellite instability (MSI) than does any other type of sporadic human cancer. The reasons for this high frequency of MSI are not yet known. In contrast to endometrial and colorectal carcinoma, mutations of the DNA mismatch repair (MMR) genes hMLH1 or hMSH2 have not been described in gastric carcinoma. However, hypermethylation of the hMLH1 MMR gene promoter is quite common in MSI-positive endometrial and colorectal cancers. This hypermethylation has been associated with hMLH1 transcriptional blockade, which is reversible with demethylation, suggesting that an epigenetic mechanism underlies hMLH1 gene inactivation and MMR deficiency. Therefore, we studied the prevalence of hMLH1 promoter hypermethylation in a total of 65 gastric tumors: 18 with frequent MSI (MSI-H), 8 with infrequent MSI (MSI-L), and 39 that were MSI negative. We found a striking association between hMLH1 promoter hypermethylation and MSI; of 18 MSI-H tumors, 14 (77.8%) showed hypermethylation, whereas 6 of 8 MSI-L tumors (75%) were hypermethylated at hMLH1. In contrast, only 1 of 39 (2.6%) MSI-negative tumors demonstrated hMLH1 hypermethylation (P<0.0001 for MSI-H or MSI-L versus MSI-negative). Moreover, hypermethylated cancers demonstrated diminished expression of hMLH1 protein by both immunohistochemistry and Western blotting, whereas nonhypermethylated tumors expressed abundant hMLH1 protein. These data indicate that hypermethylation of hMLH1 is strongly associated with MSI in gastric cancers and suggest an epigenetic mechanism by which defective MMR occurs in this group of cancers 

 ABSTRACT:

Allelic loss of chromosome 18q has been noted in intestinal type gastric adenocarcinomas. Smad4 is a gene located at 18q that was recently cloned in humans and found to be significantly altered in pancreatic cancers. We sought to determine whether Smad4 genetic alterations played a significant role in gastric tumorigenesis by studying 35 gastric adenocarcinomas of all histopathological types and pathological stages. Microdissected specimens were used for mutational analysis of Smad4 at the nucleotide level, including the entire coding region and intron/exon boundaries. Allelic imbalance was also analyzed at the Smad4 locus using two nearby microsatellite markers. One case of apparent biallelic inactivation of Smad4 was found in our study of 35 gastric carcinomas. A nonsense point mutation at codon 334 was demonstrated, which, similar to other Smad4 mutations, is predicted to truncate the conserved COOH-terminal domain of this protein. This Smad4 C to T transition mutation was proven to be somatically acquired. Allelic loss was also noted on chromosome 18q at a marker near Smad4 in this mutated gastric cancer, apparently producing complete inactivation of Smad4 in this tumor. Significant 18q allelic loss (56% of 34 informative cases) was noted in our gastric carcinomas using microsatellite markers near the Smad4 locus, regardless of histological subtype or pathological stage. Additionally, three cases of microsatellite instability were observed. Thus, Smad4 inactivation was noted in our gastric carcinomas; however, this event was rare. The frequent loss of chromosomal arm 18q observed in gastric cancers suggests the presence of other tumor suppressor genes in this region that are involved in gastric tumorigenesis. Further studies are needed to identify these other targets of inactivation during gastric cancer development.

 ABSTRACT:

The E2F group of transcription factors transactivates genes that promote progression through the G1-S transition of the cell cycle. Members of the retinoblastoma (Rb) family of proteins bind to E2Fs and inhibit this function. E2F-4, one example of the E2F group, functions as an oncogene when transfected into nontransformed cells in vitro. On the other hand, mice that are homozygously lacking a normal E2F-1 gene develop cancers, consistent with a tumor-suppressive role for this gene. The exact function of E2Fs has thus been unclear; moreover, direct involvement of this gene in primary human tumorigenesis has not been shown. We, therefore, investigated mutation within the E2F-4 coding region in 16 primary gastric adenocarcinomas, 12 ulcerative colitis-associated neoplasms, 46 sporadic colorectal carcinomas, 9 endometrial cancers, and 3 prostatic carcinomas. We limited our investigation to the serine repeat within E2F-4, reasoning that this tract might be altered in genetically unstable tumors (replication error-positive, or RER+). All tumors were RER+, with the exception of a control group of 15 RER- sporadic colorectal carcinomas. PCR with incorporation of [32P]dCTP was performed using primers flanking the serine trinucleotide (AGC) repeat. Twenty-two of 59 gastrointestinal tumors (37%) contained E2F-4 mutations; these comprised 5 of 16 gastric tumors (31%), 4 of 12 ulcerative colitis-associated neoplasms (33%, including 1 dysplastic lesion), and 13 of 31 sporadic colorectal cancers (42%). No mutation was present in any of the endometrial, prostate, or RER- colorectal tumors. Of note, homozygous mutations occurred in three cases, and two of seven informative patients showed loss of one E2F-4 allele in their tumors. Furthermore, the RER+ sporadic colorectal tumors were evaluated at trinucleotide repeats within the genes for N-cadherin and B-catenin; no tumors demonstrated mutation of these genes. These data suggest that E2F-4 is a target of defective DNA repair in these tumors.

 ABSTRACT:

Mutations within microsatellite sequences, consisting of additions or deletions of repeat units, are known as the replication/repair error positive (RER+) phenotype or micorsatellite instability (MI). Microsatellite instability has been demonstrated in hereditary and sporadic colorectal carcinomas and is usually observed in noncoding regions of genomic DNA. However, relatively few coding region targets of MI have been identified thus far. Using PCR, we amplified regions encompassing (A)8 and (C)8 microsatellite tracts within hMSH3 and hMSH6 from 31 RER+ sporadic colorectal tumors, 8 hereditary colon cancers, 23 RER+ gastric carcinomas, and 32 RER- gastric tumors. Mutations were found in 11 (36%) of 31 sporadic colon carcinomas, 4 (50%) of 8 hereditary colorectal cancers, and 5 (22%) of 23 RER+ gastric carcinomas, but in only 2 (6%) of 32 RER- gastric carcinomas. These frameshift mutations cause premature stop codons downstream that are predicted to abolish normal protein function. Our results and those of others suggest that DNA mismatch repair genes, such as hMSH3 and hMSH6, are targets for the mutagenic activity of upstream mismatch repair gene mutations and that this enhanced genomic instability may accelerate the accumulation of mutations in RER+ tumors.

 ABSTRACT:

The prominent role of the APC gene in colorectal tumor development is well established. However, its role in tumorigenesis in other tissues is not clear. Hence, DNA from 30 primary sporadic gastric adenocarcinomas was obtained from patients living in a high risk area of the world (North-Central Italy) in order to further define APC's role in gastric tumorigenesis. We thoroughly examined that region of APC which is commonly mutated in colorectal tumors using proven sensitive methods. The IVS protein assay and DNA sequence analysis of APC codons 686 through 1693 revealed no intragenic mutations. However, allelic loss of loci near APC was detected in 7 (28%) of 25 informative gastric adenocarcinomas using two 5q dinucleotide repeat markers for LOH analysis. These results suggest that genetic alteration of a region of APC commonly mutated in colorectal cancer is not a common event during sporadic gastric tumor development, at least in patients from North-Central Italy. Further analysis of chromosome 5q might identify another gene to be significantly altered in these gastric cancers.

No abstract available.