Neoplasms of the ampulla of vater with concurrent pancreatic intraductal neoplasia: a histological and molecular study.

Adenoma and adenocarcinoma of the ampulla of Vater are uncommon neoplasms of the gastrointestinal tract. Only one report has analyzed the relationship between ampullary adenocarcinoma and pancreatic intraductal neoplasia (PanIN), the precursor lesion of pancreatic adenocarcinoma. An association between PanIN and ampullary adenoma has not been reported previously. Case reports have documented the progression of PanIN to invasive pancreatic adenocarcinoma. We reviewed five resected ampullary adenoma and 17 ampullary adenocarcinoma cases and evaluated the pancreas for PanIN. Pancreatic sections from 35 autopsies were reviewed as a control group. Immunohistochemistry for overexpression of p53 and COX-2 proteins was performed in selected cases, as was PCR analysis for K-ras mutations. Follow-up clinical data were obtained. All 22 ampullary neoplasms were associated with PanIN, which was high grade in two (40%) adenoma cases and seven (41%) adenocarcinoma cases. In 16 (73%) evaluable cases, PanIN extended to the pancreatic resection margin; two of which had high grade PanIN. Among the autopsy controls eight (23%) had low-grade PanIN. Seven of the 22 ampullary cases but none of the autopsy controls had coexistent pancreatitis. A smoking history was present in two of four autopsy cases in which this history was available. Overexpression of the p53 and COX-2 proteins was present in only one case of high-grade PanIN. K-ras mutations were present in four of four of the PanIN lesions evaluated, including one autopsy case. Clinical follow-up revealed no progression of PanIN to invasive carcinoma in the remnant pancreas, although the follow-up period was too short to adequately assess that risk (an average of 3.8 y for adenoma cases and 2.5 y for adenocarcinoma cases). We conclude that adenomas and carcinomas of the ampulla are associated with PanIN, and often high-grade PanIN. Although its malignant potential has not been fully established, PanIN is underreported and often unrecognized. PanIN may be analogous to colorectal adenoma in that both are prevalent in the older adult population, but few progress to carcinoma.

The role of cyclooxygenase 2 in ulcerative colitis-associated neoplasia.

Cyclooxygenase 2 (COX-2) overexpression has been described in sporadic colonic neoplasia, but its role in ulcerative colitis (UC) neoplastic progression remains unexplored. Although the specific role of cyclooxygenase in colonic neoplasia is uncertain, its inhibition by nonsteroidal anti-inflammatory drugs decreases the risk of sporadic colonic adenocarcinoma and causes regression of adenomas in familial adenomatous polyposis. To investigate the role of COX-2 in UC-associated neoplasia, we assessed COX-2 protein and mRNA expression throughout the spectrum of UC-associated neoplastic lesions in four total colectomy specimens, using immunocytochemistry and a novel TaqMan reverse transcriptase-polymerase chain reaction assay. The findings were correlated with DNA ploidy and inflammatory activity. We found COX-2 overexpression throughout the neoplastic spectrum in UC (P: < 0.0001, R:(2)=0.53), even in diploid samples that were negative for dysplasia. Overall, neoplastic change explained 53% of the variation in COX-2 expression, whereas inflammatory activity explained only 11%. COX-2 was overexpressed in all aneuploid samples and in 38% of diploid samples (P: = 0.0074). cDNA representational difference analysis was also performed and revealed that COX-2 mRNA was an up-regulated cDNA representational difference analysis difference product. COX-2 overexpression occurs early in UC-associated neoplasia, and the increase cannot be explained by inflammatory activity alone. The data suggest that COX-2-specific inhibitors may have a chemopreventative role in UC but the possibility that they could exacerbate UC inflammatory activity needs to be tested.

Pancolonic chromosomal instability precedes dysplasia and cancer in ulcerative colitis.

Patients with long-standing ulcerative colitis (UC) are at increased risk for colon cancer. These cancers are thought to arise from preexisting dysplasia in a field of abnormal cells that often exhibits aneuploidy and p53 abnormalities. Using dual color fluorescence in situ hybridization with centromere probes and locus-specific arm probes for chromosomes 8, 11, 17, and 18, we demonstrate that chromosomal instability (CIN) is present throughout the colon of UC patients with high-grade dysplasia or cancer. In rectal biopsies that were negative for dysplasia, abnormalities in chromosomal arms, especially losses, were most common, whereas centromere gains were most common in dysplasia and cancer. The frequency and type of abnormalities varied between the chromosomes examined; chromosome 8 was the least affected, and 17p loss was found to be an early and frequent event. Chromosomal arm instability showed 100% sensitivity and specificity for distinguishing control biopsies from histologically negative rectal biopsies from these UC patients, raising the possibility that a screen for CIN might detect the subset of UC patients who are at greatest risk for development of dysplasia and cancer. These results suggest that dysplasia and cancer in UC arise from a process of CIN that affects the entire colon; this may provide the mutator phenotype that predisposes to loss of tumor suppressor genes and evolution of cancer.

Microsatellite instability in nonneoplastic mucosa from patients with chronic ulcerative colitis.

Microsatellite instability (MIN) has been detected in many cancer types; however, recently we also observed it in the nonneoplastic but inflammatory setting of pancreatitis. Consequently, we sought to examine whether MIN was present in another inflammatory condition, ulcerative colitis (UC). MIN was found in 50% of UC patients whose colonic mucosa was negative for dysplasia, 46% of those with high-grade dysplasia, and 40% of those with cancer but in none of the ischemic or infectious colitis controls (P<0.03). Thus, UC patients may have MIN within mucosa that has no histological evidence of neoplastic change. MIN in this setting may reflect the inability of DNA repair mechanisms to compensate for the stress of chronic inflammation, and may be one mechanism for the heightened neoplastic risk in UC.

Risk and natural history of colonic neoplasia in patients with primary sclerosing cholangitis and ulcerative colitis.

BACKGROUND & AIMS: Primary sclerosing cholangitis (PSC) has been suggested as a risk factor for the development of colorectal cancer in ulcerative colitis (UC); however, previous studies of this association have been limited by small numbers of patients with PSC or have been performed retrospectively. This study prospectively evaluates the risk and natural history of colonic tumorigenesis in patients with PSC and UC and compares it with patients with UC without PSC. METHODS: Twenty patients with PSC and UC and 25 control patients with UC were followed prospectively by colonoscopic surveillance using extensive mucosal biopsy sampling. All control patients with UC had disease extending beyond the sigmoid colon of > or = 8 years' duration; patients with PSC and UC were studied regardless of disease duration. RESULTS: Forty-five percent (9 of 20) of the patients with PSC and UC had dysplasia compared with 16% (4 of 25) of the control patients with UC (P < or = 0.002). Prior liver transplantation did not affect the risk of colonic dysplasia. The time course for progression to dysplasia was similar between the patients with PSC and UC and the patients with UC; however, the patients with PSC and UC were five times more likely to develop dysplasia. CONCLUSIONS: Patients with PSC and UC represent a subset of patients with UC who are at markedly increased risk for colonic neoplasia and who need close colonoscopic surveillance with extensive biopsy sampling.

A germline substitution in the human MSH2 gene is associated with high-grade dysplasia and cancer in ulcerative colitis.

BACKGROUND & AIMS: The DNA mismatch repair gene human MSH2 shows a germline mutation in certain family members with hereditary nonpolyposis colorectal cancer. There is an increased risk of colorectal cancer in patients with ulcerative colitis (UC) with extensive disease of > 8 years' duration; however, specific constitutional predisposing genetic abnormalities have not yet been identified. METHODS: A germline human MSH2 abnormality was sought in patients with UC with high-grade dysplasia or carcinoma. RESULTS: After direct sequencing of exon 13 and flanking regions of human MSH2, a germline T to C substitution was shown at the -6 intronic splice acceptor site of exon 13. This substitution was found in 14 of 53 patients with UC with high-grade dysplasia or carcinoma (26%) compared with 4 of 36 high-risk patients with UC without dysplasia or cancer (11%) (P < or = 0.04) and in 7 of 80 healthy adult blood donors (9%) (P < or = 0.003). The patients with UC who had the substitution were three times more likely to develop neoplasia than patients with UC who did not carry it. CONCLUSIONS: An intronic splice-site substitution in the human MSH2 gene is present in the general population but may predispose to cancer in the setting of UC.

Mutations in the p53 gene: an early marker of neoplastic progression in ulcerative colitis.

BACKGROUND/AIMS: In long-term extensive ulcerative colitis, aneuploidy occurs earlier and loss of heterozygosity for p53 (p53 LOH) later during histological progression towards carcinoma. This study determined the time of onset of p53 mutation in this progression. METHODS: We developed a rapid, sensitive screening assay for p53 mutations at codon 248. The geographic distribution of this p53 mutation was mapped in two fresh colectomy specimens with mutations of codon 248 (1 cancer, 1 dysplasia) and correlated with patterns of clonal expansion, histological progression, and allelic loss. Numerous samples from throughout both colons were analyzed (216 for histology, 142 for DNA content, 104 for mutation, and 41 for p53 LOH). RESULTS: p53 mutation correlated highly with histological grade and was distributed more extensively than p53 LOH. Mutation, but not LOH, was also found in diploid, nondysplastic colonic mucosa adjacent to dysplastic areas. CONCLUSIONS: These findings suggest that p53 mutation appears to be an early genetic event that precedes p53 LOH. The very close correlation of p53 mutation with aneuploidy (P > 0.0001) emphasizes the role of normal p53 at the G1 checkpoint to help prevent entry of genetically damaged cells into the cell cycle.

Neoplastic progression in ulcerative colitis: histology, DNA content, and loss of a p53 allele.

Neoplastic progression in patients with chronic ulcerative colitis (UC) is characterized by the development of epithelial dysplasia, which is accompanied by genetic abnormalities that can be detected by flow cytometric and molecular biologic methods. Distribution of and correlation between histologic abnormalities, DNA content, and loss of heterozygosity for a p53 allele (p53 LOH) in the colons of nine UC patients were analyzed. Loss of a p53 allele was found in 85% (22/26) of biopsy specimens classified histologically as carcinoma, 63% (25/40) of biopsy specimens with high grade dysplasia, and 33% (7/21) of biopsy specimens with low grade dysplasia. Loss of heterozygosity for p53 was also found in 9% (5/57) of biopsy specimens indefinite for dysplasia and in 1/18 biopsy specimens negative for dysplasia, showing that this genetic change may occur early in the histological progression towards carcinoma. Aneuploid DNA contents were more common than p53 LOH in regions with negative, indefinite or low grade dysplastic histology; moreover, p53 LOH was detected only in aneuploid cells and not in diploid epithelium. Aneuploidy alone was not as specific a marker for the concomitant presence of dysplasia or carcinoma in a biopsy sample as aneuploidy combined with p53 LOH. These findings show that aneuploidy may precede both p53 LOH and epithelial dysplasia. Two UC patients' colons contained geographically separated clones of cells with different aneuploidies that also showed loss of different p53 alleles, suggesting that neoplasia may arise within different populations of cells in separate areas of the same colon.

Evolution of the salmonid mitochondrial control region.

To explore the evolutionary nature of the salmonid mitochondrial DNA (mtDNA) control region (D-loop) and its utility for inferring phylogenies, the entire region was sequenced from all eight species of anadromous Pacific salmon, genus Oncorhynchus; the Atlantic salmon, Salmo salar; and the Arctic grayling, Thymallus arcticus. A comparison of aligned sequences demonstrates that the generally conserved sequence elements that have been previously reported for other vertebrates are maintained in these primitive teleost fishes. Results reveal a significantly nonrandom distribution of nucleotide substitutions, insertions, and deletions that suggests that portions of the salmonid D-loop may be under differential selective constraints and that most of the control region of these fishes may evolve at a rate similar to that of the remainder of their mtDNA genomes. Maximum likelihood and Fitch parsimony analyses of 9 kb of aligned salmonid sequence data give evolutionary trees of identical topology. These results are consistent with previous molecular studies of a limited number of salmonid taxa and with more comprehensive, classical analyses of salmonid evolution. Predictions from these data, based on a molecular clock assumption for the mtDNA control region, are also consistent with fossil evidence that suggests that species of Oncorhynchus could be as old as the Middle Pliocene and would have thus given rise to the extant Pacific salmon prior to about 5 or 6 million years ago.

Frequent loss of a p53 allele in carcinomas and their precursors in ulcerative colitis.

Allelic deletions of the p53 gene previously were demonstrated by Southern hybridization to occur in high frequency in sporadic colon carcinomas and in a variety of other human tumors. We have examined the frequency of allelic loss of the p53 gene in carcinoma and dysplasia arising in patients with chronic ulcerative colitis who are heterozygous for the codon 72 polymorphism in exon 4 of the p53 gene. Cells derived from carcinoma and dysplasia specimens from 10 patients who were heterozygous at this locus were sorted by flow cytometry on the basis of DNA content. The p53 exon 4 region was amplified from diploid and aneuploid populations, via a polymerase chain reaction (PCR), and digested with BstUI. Three of three carcinomas, four of six dysplasias, and one patient who was indefinite for dysplasia demonstrated evidence of allelic loss of the p53 gene. Seven of ten cases of sporadic colon carcinoma, analyzed for comparative purposes, exhibited loss of a p53 allele. These results demonstrate that PCR analysis, followed by restriction endonuclease digestion of a polymorphic locus, can provide a rapid, definitive method for analyzing loss of heterozygosity in small numbers of cells from colonic mucosa. Such loss precedes cancer in ulcerative colitis and can be present in its earliest histologically identifiable precursor.