Comparative molecular pathology of sporadic hyperplastic polyps and neoplastic lesions from the same individual.

AIM: The biology of colorectal hyperplastic polyps is of considerable relevance, because recent evidence suggests that under certain circumstances hyperplastic polyps may be precursors of neoplasms. The aim of this study was to assess and compare the clinical and molecular characteristics of hyperplastic polyps and neoplastic lesions removed from patients without the hyperplastic polyposis syndrome. METHODS: One hundred and twenty six patients were identified through a series of genetic epidemiological studies. Each patient had at least one neoplastic lesion and one hyperplastic polyp; there was a total of 147 hyperplastic polyps. All lesions were evaluated for K-ras mutations, loss of heterozygosity (LOH) of the adenomatous polyposis coli (APC) gene, and microsatellite instability. RESULTS: K-ras mutation was detected in 15 (10%) hyperplastic polyps, all from the rectosigmoid colon. No hyperplastic polyp had APC LOH or microsatellite instability. Patients with adenomas or carcinomas showing K-ras mutations were not more likely to have hyperplastic polyps with K-ras mutations. The average number of adenomas did not differ between those patients with hyperplastic polyps with K-ras mutations and those without K-ras mutations. There was no association between the hyperplastic polyp and the adenoma regarding the colon segments from which the two lesions were removed. CONCLUSIONS: The sporadic hyperplastic polyp is a lesion with limited molecular change and no relation to patients' neoplastic lesions.

Molecular changes in the Ki-ras and APC genes in primary colorectal carcinoma and synchronous metastases compared with the findings in accompanying adenomas.

AIMS: To compare the molecular genetic changes in the Ki-ras and adenomatous polyposis coli (APC) genes between colorectal carcinomas and synchronous metastases, and then to compare and contrast those changes with previously reported changes in the two genes between these carcinomas and accompanying adenomas. This expanded comparison would provide greater understanding of the progression of molecular changes in neoplastic tissue during the development of malignancy from a benign adenoma to carcinoma and then to metastatic spread of the malignancy. METHODS: DNA was extracted from paraffin wax embedded tissue. This was followed by polymerase chain reaction and gel electrophoresis for mutations in the Ki-ras gene using single stranded conformational polymorphism analysis. Amplification of a CA repeat marker was used to assess loss of heterozygosity (LOH) at the APC gene. RESULTS: The findings for the Ki-ras gene in 42 paired carcinomas and synchronous metastases were identical, regardless of whether or not the carcinoma and its companion adenoma had identical Ki-ras findings. The results of APC LOH for 39 paired carcinomas and synchronous metastases were also identical, whether or not the carcinoma and its companion adenoma had identical APC LOH findings. Results were uninformative for three pairs. CONCLUSIONS: With respect to these two genes, a carcinoma may be discordant from its companion adenoma, but the metastasis remains consistent with the colonic carcinoma.

Molecular changes in the Ki-ras and APC genes in colorectal adenomas and carcinomas arising in the same patient.

The purpose of this study was to compare the molecular genetic changes in the Ki-ras and adenomatous polyposis coli (APC) genes between adenomas and carcinomas removed from the same patients. This comparison of benign and malignant tissue would enhance understanding of the progression of molecular changes during the development of colorectal malignancy and similarities between paired lesions could be indicative of a common aetiology. The basic procedures used were DNA extraction from wax blocks of removed tissue, followed by polymerase chain reaction (PCR) and gel electrophoresis for mutations in the Ki-ras gene using single strand conformational polymorphism (SSCP); amplification of a CA repeat marker was used to assess for loss of heterozygosity (LOH) of the APC gene. The main findings in 100 adenoma and carcinoma pairs for the Ki-ras gene were as follows: the frequency of Ki-ras mutation in the adenomas increased with increasing villous component, but did not vary in the paired carcinomas; the frequency of Ki-ras mutation in villous adenomas was greater than in carcinomas; and when both paired lesions had Ki-ras mutations, only 44% had the identical mutation. For the APC gene, the incidence of LOH in the adenomas did not vary by histological type; the LOH status of the adenoma was associated with that of the paired carcinoma; but when both paired lesions had LOH of the APC gene, only 50% had LOH for the same allele. In conclusion, these data on paired adenomas and carcinomas suggest that a Ki-ras mutation is not a consistent finding between the adenoma and carcinoma from the same bowel. The development of LOH of the APC gene is a slightly more consistent finding between the pair, but is not always allelic-specific.

Molecular evidence that the stromal and epithelial cells in pleomorphic adenomas of salivary gland arise from the same origin: clonal analysis using human androgen receptor gene (HUMARA) assay.

Salivary gland pleomorphic adenomas are characterized by a biphasic growth of "epithelial" and "stromal" regions. The "epithelial" region is a compactly organized mixture of both luminal and nonluminal cells, whereas the stromal region is composed predominantly of the nonluminal cells. Using the polymerase chain reaction (PCR)-based HUMARA assay on DNA from formalin-fixed, paraffin-embedded tissues from pleomorphic adnomas of female patients, we intend to clarify the clonal relation between the luminal and nonluminal cells and the clonal nature of the morphologically diverse nonluminal cells in this tumor. HUMARA, the human androgen receptor gene, is located on the X chromosome and contains a segment of polymorphic CAG tandem repeats in exon 1. Several methylation-sensitive HhaI restriction sites are located 5' to these CAG repeats. It is an ideal tool to study clonality of female tissues by examining the methylation pattern. Of the 13 cases analyzed, 3 were homozygous at the HUMARA locus and therefore noninformative. The remaining 10 cases were informative. All 10 cases showed a monoclonal pattern in the stromal area, indicating that the morphologically diverse nonluminal cells are monoclonal. Eight of the 10 cases showed monoclonality in the "epithelial" areas, suggesting a common clonality between luminal and nonluminal cells. Of the remaining 2 samples, 1 was polyclonal for the "epithelial" region, and the other was not amplifiable. Our data provide the first molecular evidence that the luminal and nonluminal cells in pleomorphic adenomas arise from the same clone in most cases, and the morphologically diverse nonluminal cells are monoclonal.

K-ras mutation and loss of heterozygosity of the adenomatous polyposis coli gene in patients with colorectal adenomas with in situ carcinoma.

BACKGROUND: The majority of colorectal carcinomas, if not all, arise from a benign adenoma. The DNA of the carcinomatous cells frequently has mutations in several genes. However, it is not exactly clear when during the neoplastic process each mutation develops. An adenoma with an area of in situ carcinoma provides an opportunity to evaluate genetic changes within a single neoplasia whose separate areas are comprised of both the benign adenoma as well as the malignant carcinoma. METHODS: Thirty-seven neoplasms with areas of both benign adenoma and in situ carcinoma were studied. Both portions were evaluated for loss of heterozygosity (LOH) of the adenomatous polyposis coli (APC) gene and for mutations in codons 12/13 of the K-ras oncogene using the polymerase chain reaction technique. RESULTS: Twenty-eight neoplasms showed no LOH in either portion whereas both portions of 4 neoplasms revealed a loss of heterozygosity. In three lesions the APC gene was normal in the adenomatous portion but LOH was present in the carcinomatous portion. Two neoplasms were uninformative for LOH of the APC gene. Thirteen neoplasms showed the wild-type pattern for the K-ras oncogene whereas 15 contained the identical mutation in both portions. Of the remaining nine neoplasms, six had a K-ras mutation in the adenomatous portion only and three had one pattern in the adenomatous portion and a different pattern in the in situ carcinoma portion. CONCLUSIONS: LOH of the APC gene is an early and persistent feature in the evolution of a benign colorectal adenoma into an in situ carcinoma. There is less consistency regarding K-ras mutations; one in five in situ carcinomas contains a K-ras mutation different from that observed in the adenomatous portion.

Comparison of allelic ratios from paired blood and paraffin-embedded normal tissue for use in a polymerase chain reaction to assess loss of heterozygosity.

BACKGROUND: One method to assess loss of heterozygosity (LOH) of various genes is the amplification of DNA from neoplastic tissue by using microsatellite markers. LOH can best be considered on a quantitative basis as a comparison of allelic ratios of neoplastic tissue to that of the normal control. We will illustrate through quantitative methods the importance of using the appropriate controls when determining allelic loss. METHODS AND RESULTS: DNA extracted from 28 paired blood and formalin-fixed, paraffin-embedded normal mucosal tissue was amplified using the DP1 microsatellite marker, consisting of a variable number of CA repeats. This marker is located within the D5S346 (DP1) region on chromosome 5 and is linked to the adenomatous polyposis coli gene. Allelic ratios were calculated after scanning autoradiographs on a densitometer. Ratio values approaching 1 were observed when the two alleles were close in molecular weight, whereas ratios less than 1 were detected when the two alleles had very different molecular weights. This discrepancy was more pronounced in paraffin-embedded tissue than with blood samples. CONCLUSION: For LOH amplification assays, it is best to use normal control samples that are of the same tissue source as the neoplastic sample being analyzed. When assessing LOH in neoplastic tissue, a quantitative value rather than visual assessment of the alleles should be considered. The values may be normalized by dividing the ratio of the two tumor alleles by the ratio of the two normal alleles.

Factor V Leiden mutation is not increased in patients with inflammatory bowel disease.

Individuals with inflammatory bowel disease (IBD) are known to have an increased incidence of thromboembolic disease. Activated protein C resistance (APCR) has been identified as one of several inherited disorders of coagulation that predispose individuals to thromboembolic problems. This resistance results from a single point mutation in the factor V gene, called factor V Leiden. It has been suggested that many patients with IBD have APCR, as tested by a clotting assay. We have evaluated a series of 49 patients with IBD, none of whom had a history of thromboembolic disease. We assayed for the factor V Leiden mutation by polymerase chain reaction and found only one heterozygote. Seventeen of the 49 patients were negative for APCR by the clotting assay. Factor V Leiden mutation is not more common in patients with IBD than in the general population. We were unable to confirm a prior report indicating that patients with IBD have a higher prevalence of resistance to activated protein C.