Evolution of neoplastic cell lineages in Barrett oesophagus.

It has been hypothesized that neoplastic progression develops as a consequence of an acquired genetic instability and the subsequent evolution of clonal populations with accumulated genetic errors. Accordingly, human cancers and some premalignant lesions contain multiple genetic abnormalities not present in the normal tissues from which the neoplasms arose. Barrett oesophagus (BE) is a premalignant condition which predisposes to oesophageal adenocarcinoma (EA) that can be biopsied prospectively over time because endoscopic surveillance is recommended for early detection of cancer. In addition, oesophagectomy specimens frequently contain the premalignant epithelium from which the cancer arose. Neoplastic progression in BE is associated with alterations in TP53 (also known as p53) and CDKN2A (also known as p16) and non-random losses of heterozygosity (LOH). Aneuploid or increased 4N populations occur in more than 90-95% of EAs, arise in premalignant epithelium and predict progression. We have previously shown in small numbers of patients that disruption of TP53 and CDKN2A typically occurs before aneuploidy and cancer. Here, we determine the evolutionary relationships of non-random LOH, TP53 and CDKN2A mutations, CDKN2A CpG-island methylation and ploidy during neoplastic progression. Diploid cell progenitors with somatic genetic or epigenetic abnormalities in TP53 and CDKN2A were capable of clonal expansion, spreading to large regions of oesophageal mucosa. The subsequent evolution of neoplastic progeny frequently involved bifurcations and LOH at 5q, 13q and 18q that occurred in no obligate order relative to each other, DNA-content aneuploidy or cancer. Our results indicate that clonal evolution is more complex than predicted by linear models.

Regulation of mating in the cell cycle of Saccharomyces cerevisiae.

The capacity of haploid a yeast cells to mate (fuse with a haploid strain of alpha mating type followed by nuclear fusion to produce a diploid cell) was assessed for a variety of temperature-sensitive cell division cycle (cdc) mutants at the permissive and restrictive temperatures. Asynchronous populations of some mutants do not mate at the restrictive temperature, and these mutants define genes (cdc 1, 4, 24, and 33) that are essential both for the cell cycle and for mating. For most cdc mutants, asynchronous populations mate well at the restrictive temperature while populations synchronized at the cdc block do not. Populations of a mutant carrying the cdc 28 mutation mate well at the restrictive temperature after synchronization at the cdc 28 step. These results suggest that mating can occur from the cdc 28 step, the same step at which mating factors arrest cell cycle progress. The cell cycle interval in which mating can occur may or may not extend to the immediately succeeding and diverging steps (cdc 4 and cdc 24). High frequency mating does not occur in the interval of the cell cycle extending from the step before the initiation of DNA synthesis (cdc 7) through DNA synthesis (cdc 2, 8, and 21), medial nuclear division (cdc 13), and late nuclear division (cdc 14 and 15).

A p53-dependent mouse spindle checkpoint.

Cell cycle checkpoints enhance genetic fidelity by causing arrest at specific stages of the cell cycle when previous events have not been completed. The tumor suppressor p53 has been implicated in a G1 checkpoint. To investigate whether p53 also participates in a mitotic checkpoint, cultured fibroblasts from p53-deficient mouse embryos were exposed to spindle inhibitors. The fibroblasts underwent multiple rounds of DNA synthesis without completing chromosome segregation, thus forming tetraploid and octaploid cells. Deficiency of p53 was also associated with the development of tetraploidy in vivo. These results suggest that murine p53 is a component of a spindle checkpoint that ensures the maintenance of diploidy.

Progressive region-specific de novo methylation of the p16 CpG island in primary human mammary epithelial cell strains during escape from M(0) growth arrest.

CpG island methylation plays an important role in normal cellular processes, such as genomic imprinting and X-chromosome inactivation, as well as in abnormal processes, such as neoplasia. However, the dynamics of de novo CpG island methylation, during which a CpG island is converted from an unmethylated, active state to a densely methylated, inactive state, are largely unknown. It is unclear whether the development of de novo CpG island methylation is a progressive process, in which a subset of CpG sites are initially methylated with a subsequent increase in methylation density, or a single event, in which the initial methylation event encompasses the entire CpG island. The tumor suppressor gene p16/CDKN2a/INK4a (p16) is inactivated by CpG island methylation during neoplastic progression in a variety of human cancers. We investigated the development of methylation in the p16 CpG island in primary human mammary epithelial cell strains during escape from mortality stage 0 (M(0)) growth arrest. The methylation status of 47 CpG sites in the p16 CpG island on individual DNA molecules was determined by sequencing PCR clones of bisulfite-treated genomic DNA. The p16 CpG island was initially methylated at a subset of sites in three discrete regions in association with p16 transcriptional repression and escape from M(0) growth arrest. With continued passage, methylation gradually increased in density and methylation expanded to sites in adjacent regions. Thus, de novo methylation in the p16 CpG island is a progressive process that is neither site specific nor completely random but instead is region specific. Our results suggest that early detection of methylation in the CpG island of the p16 gene will require methylation analysis of the three regions and that the identification of region-specific methylation patterns in other genes may be essential for an accurate assessment of methylation-mediated transcriptional silencing.

The influence of a NAPL on the loss and biodegradation of 14C-phenanthrene residues in two dissimilar soils.

This study was carried out to assess the influence of diesel, applied over a log concentration range, on the loss and extractability of phenanthrene (measured as putative 14C-phenanthrene residues) in two different soils. The influence of diesel on the ability of a cyclodextrin based extraction method to predict the microbial bioavailability of 14C-residues was also assessed. An increase in loss of 14C-residues with increasing diesel concentration from 0 to 2000 mg kg-1 was generally observed with time in both soils. It is suggested that this trend is attributable to competitive sorption for soil sorption sites and to a lesser extent to displacement of 14C-residues from soil sorption sites by diesel resulting in greater compound availability and therefore greater loss by degradation via the actions of indigenous microorganisms. However, in the 20000 mg kg-1 diesel treatments of both soils, results indicated a delayed loss. It is suggested that this retarded loss was due to the formation of a discrete NAPL-phase into which 14C-phenanthrene residues partitioned, thereby decreasing their availability and as a consequence their degradation. Furthermore, it is suggested that nutrient limitation may have slowed down degradation rates as diesel concentrations increased. Comparison between cyclodextrin-extractability and microbial mineralisation supported the use of cyclodextrin to assess microbial bioavailability of 14C-residues after 50 d or more ageing up to diesel concentrations of 2000 mg kg-1. However, results suggested that at high diesel concentrations (specifically 20000 mg kg-1) co-extraction of 14C-phenanthrene residues may have occurred as a result of the combined solvation powers of both the cyclodextrin and the diesel. Furthermore, mineralisation of 14C-phenanthrene residues may have been affected by extreme nutrient limitation in this treatment.

Comparison of selected non-exhaustive extraction techniques to assess PAH availability in dissimilar soils.

Recently, it has become apparent that the use of total contaminant concentrations as a measure of potential contaminant exposure to plants or soil organisms is inappropriate and that bioavailability of contaminants is a better measure of potential exposure. In light of this, non-exhaustive extraction techniques are being investigated to assess their appropriateness in determining bioavailability. In this study, phenanthrene extractability using hydroxypropyl-beta-cyclodextrin (HPCD) and desorption kinetics using butan-1-ol (BuOH) were determined in three dissimilar spiked soils. The soils were extracted after 1 d, 40 d and 80 d of soil-compound contact time. The amount of phenanthrene extracted by HPCD was compared to the rapidly desorbed fraction removed by BuOH. Further experiments using the same soils and extraction methods to assess the relative extractability of phenanthrene, pyrene and benzo(a)pyrene were conducted. Overall, the extraction methods used in this study had different extraction efficiencies. Results suggest that as compound hydrophobicity increased, BuOH became a more exhaustive extractant with respect to HPCD, especially for soils with high clay and organic matter content. These results are important as they highlight differences between two contrasting non-exhaustive extraction techniques both of which have been suggested to be appropriate in the assessment of bioavailability.

Clonal expansion and loss of heterozygosity at chromosomes 9p and 17p in premalignant esophageal (Barrett's) tissue.

BACKGROUND: Abnormalities involving the p16 (also known as cyclin-dependent kinase N2 [CDKN2], p16 [INK4a], or MTS1) and p53 (also known as TP53) tumor suppressor genes are highly prevalent in esophageal adenocarcinomas. Loss of heterozygosity (LOH) at 9p21 and 17p13 chromosomes (locations for p16 and p53 genes, respectively) is frequently observed in the premalignant condition, Barrett's esophagus. We studied extensively the distribution and heterogeneity of LOH at 9p and 17p chromosomes throughout the Barrett's segment in patients who have not yet developed esophageal adenocarcinoma. METHODS: We evaluated 404 samples from 61 consecutive patients enrolled in the Seattle Barrett's Esophagus Study from February 1995 through September 1998. All patients had high-grade dysplasia but no diagnosis of cancer. The samples were assayed for LOH at 9p and 17p chromosomes after amplification of genomic DNA by use of polymerase chain reaction and DNA genotyping. The cell fractions were purified by flow cytometry on the basis of DNA content and proliferation-associated antigen labeling. Association between LOH at 9p and LOH at 17p with flow cytometric abnormalities was determined by chi-squared test, and logistic regression models were used to model and test for the extent to which a particular genotype was found in 2-cm intervals. RESULTS AND CONCLUSIONS: LOH at 9p and 17p chromosomes are highly prevalent somatic genetic lesions in premalignant Barrett's tissue. LOH at 9p is more common than LOH at 17p in diploid samples and can be detected over greater regions of Barrett's epithelium. In most patients with high-grade dysplasia, the Barrett's mucosa contains a mosaic of clones and subclones with different patterns of LOH. Some clones had expanded to involve extensive regions of Barrett's epithelium. LOH at 9p and 17p chromosomes may be useful biomarkers to stratify patients' risk of progression to esophageal cancer.

Allelic loss and mutational analysis of the DPC4 gene in esophageal adenocarcinoma.

DPC4, a recently cloned gene located on 18q2l.l, is inactivated in almost one half of pancreatic adenocarcinomas. To determine whether DPC4 inactivation is involved in esophageal adenocarcinoma, we have analyzed aneuploid populations from biopsies of 35 patients with Barrett's esophagus who had premalignant epithelium, adenocarcinoma, or both. Sixteen of 35 patients (46%) had allelic loss at l8q21.1, including 7 patients who had only premalignant tissue present in their Barrett segment. In addition, three of four patients (75%) with l8q21.1 loss in their aneuploid populations had the allelic loss present in diploid cells. Mutational analysis of DPC4 did not reveal any inactivating alterations in the gene. These data indicate that allelic losses at l8q are selected during neoplastic progression in Barrett's esophagus, but the targeted gene remains to be identified.

p53-mutant clones and field effects in Barrett's esophagus.

Previous studies have demonstrated multifocal neoplasia in Barrett's esophagus. We evaluated 213 mapped, flow-purified, endoscopic biopsies to determine the distribution of p53-mutant clones in the Barrett's segments of 58 patients who had high-grade dysplasia without cancer. Twenty-nine patients (50%) had p53 mutations in their Barrett's segments, including 3 patients with multiple distinct p53 mutations. p53-mutant clones, including diploid cell populations, underwent expansion from 1 to 9 cm in the Barrett's segment. In 12 of 29 patients (41%) with a p53 mutation, the same mutation was found at every evaluated level of the metaplastic epithelium. This extensive p53-mutant clonal expansion suggests a somatic genetic basis for previous observations of field effects in Barrett's esophagus.

Persistent clonal areas and clonal expansion in Barrett's esophagus.

Three patients with Barrett's esophagus who had cytogenetic abnormalities detected in their metaplastic epithelium developed high-grade dysplasia or adenocarcinoma during prospective surveillance over a period of 1.5 to 6 years. In the 3 cases, cytogenetic abnormalities that were associated with the most advanced histological lesions were present in samples obtained 11, 25, and 48 months prior to the diagnosis of high-grade dysplasia or carcinoma. In a fourth patient, marker chromosomes found in a Barrett's adenocarcinoma were also present in an esophageal region spatially removed from the tumor. In all four patients, clonal cytogenetic abnormalities were present in samples obtained at widespread locations in the Barrett's segment. These observations suggest that in some patients with Barrett's esophagus clonal proliferations arise in regions of benign histology and spread to involve large areas of Barrett's mucosa. These clones persisted when the disease progressed to high-grade dysplasia or adenocarcinoma.

p16INK4a promoter is hypermethylated at a high frequency in esophageal adenocarcinomas.

Loss of heterozygosity (LOH) of 9p21, which contains the p16INK4a tumor suppressor gene locus, is one of the most frequent genetic abnormalities in human neoplasia, including esophageal adenocarcinomas. Only a minority of Barrett's adenocarcinomas with 9p21 LOH have a somatic mutation in the remaining p16 allele, and none have been found to have homozygous deletions. To determine whether p16 promoter hypermethylation may be an alternative mechanism for p16 inactivation in esophageal adenocarcinomas, we examined the methylation status of the p16 promoter in flow-sorted aneuploid cell populations from 21 patients with premalignant Barrett's epithelium or esophageal adenocarcinoma. Using bisulfite modification, primer-extension preamplification, and methylation-specific PCR, we demonstrate that the methylation assay can be performed on 2 ng of DNA (approximately 275 cells). Eight of 21 patients (38%) had p16 promoter hypermethylation and 9p21 LOH, including 3 patients who had only premalignant Barrett's epithelium. Our data suggest that promoter hypermethylation with LOH is a common mechanism for inactivation of p16 in the pathogenesis of esophageal adenocarcinomas.

Microsatellite instability occurs frequently and in both diploid and aneuploid cell populations of Barrett's-associated esophageal adenocarcinomas.

Alterations of microsatellites consisting of extra or missing copies of these sequences occur at relatively high frequencies in sporadic and hereditary colorectal adenocarcinomas, gastric and pancreatic cancers, and at lower frequencies in endometrial, bladder, ovarian, and other carcinomas. We determined the prevalence of microsatellite instability in esophageal adenocarcinoma, Barrett's esophagus, and squamous cell carcinoma of the esophagus. Assays were performed on 105 patients, including 28 subjects with Barrett's metaplasia, 36 with Barrett's-associated adenocarcinoma, and 42 with primary esophageal squamous cell carcinoma. Flow cytometric nuclear sorting based on DNA content was performed on 25 of the adenocarcinomas prior to DNA extraction. Specimens from 11 of the 106 patients (10%) showed instability at 1 or more chromosomal loci. Instability was seen in 2 of 28 patients (7%) with Barrett's metaplasia alone, in 8 of 36 (22%) with adenocarcinoma, and in 1 of 42 (2%) with squamous cell carcinoma. Among the 25 flow cytometrically sorted adenocarcinomas, instability occurred in 8 (32%); sorted diploid nuclei from these tumors showed instability in 4 of 8 cases (50%). These data indicate that microsatellite instability occurs frequently in Barrett's-associated esophageal adenocarcinoma. They also suggest that in esophageal adenocarcinomas, microsatellite instability can develop as an early event in metaplasia and in diploid tumor cells, before aneuploidy occurs.

17p allelic deletions and p53 protein overexpression in Barrett's adenocarcinoma.

Barrett's esophagus is a condition in which the stratified squamous epithelium of the esophagus is replaced by metaplastic columnar epithelium that predisposes to the development of esophageal adenocarcinoma. Allelic deletions of 17p and alterations of p53 including elevated p53 protein levels have been observed in many different tumors. To investigate the presence of 17p allelic deletions and p53 protein overexpression in Barrett's adenocarcinomas, we have combined the use of restriction fragment length polymorphism analysis, multiparameter flow cytometry, and DNA content cell sorting. The combined use of these methodologies permits the purification of aneuploid tumor cells for restriction fragment length polymorphism analysis of 17p allelic deletions and the evaluation of p53 protein expression by multiparameter flow cytometry in the same aneuploid tumor cell populations. We analyzed 15 aneuploid populations and one tetraploid populations from 13 Barrett's adenocarcinomas for 17p allelic deletions and p53 protein overexpression to determine whether both of these alterations are involved in carcinogenesis in Barrett's esophagus. Twelve of 13 tumors (92%) had 17p allelic deletions, and 8 of 13 tumors (62%) had p53 protein overexpression. Eight of the 12 tumors (67%) with 17p allelic deletions also had p53 protein overexpression. These data indicate that both 17p allelic deletions and p53 protein overexpression are frequently involved in carcinogenesis in Barrett's esophagus.

17p allelic losses in diploid cells of patients with Barrett's esophagus who develop aneuploidy.

Inactivation of the p53 gene, located on chromosome 17p, leads to genetic instability and aneuploidy in vitro. Aneuploid cell populations from Barrett's adenocarcinomas have a high prevalence of 17p allelic losses, and there is substantial evidence that the target of these losses is the p53 gene. If 17p allelic losses lead to aneuploidy in Barrett's esophagus, then they should be present in diploid cells from patients who develop aneuploidy. We detected 17p allelic losses in diploid cells from 10 of 11 patients (91%) with Barrett's esophagus who developed aneuploid cell populations. Our data strongly suggest that 17p allelic losses precede the development of aneuploidy during neoplastic progression in Barrett's esophagus in vivo and, therefore, support in vitro evidence for the role of p53 in genetic instability.

p16(INK4a) lesions are common, early abnormalities that undergo clonal expansion in Barrett's metaplastic epithelium.

Barrett's esophagus (BE) is the only known precursor to esophageal adenocarcinoma, a cancer of which the incidence has been increasing at an alarming rate in Western countries. p16(INK4a) lesions occur frequently in esophageal adenocarcinomas but their role in neoplastic progression is not well understood. We detected 9p21 loss of heterozygosity, p16 CpG island methylation, and p16 mutations in biopsies from 57%, 61%, and 15%, respectively, of 107 patients with BE. In contrast, no mutations were found in p14(ARF) or p15, and methylation was found in only 4% and 13%, respectively. >85% of Barrett's segments had clones with one (p16+/-) or two (p16-/-) p16 lesions. Both p16+/- and p16-/- clones underwent extensive expansion involving up to 17 cm of esophageal mucosa. The prevalence of established biomarkers in BE, such as 17p (p53) loss of heterozygosity, aneuploidy, and/or increased 4N (tetraploid) populations, increased from 0% to 20% to 44% in patients whose biopsies were p16+/+, p16+/-, and p16-/-, respectively (P < 0.001). Barrett's segment lengths also increased with change in p16 status with a median of 1.5, 6.0, and 8.0 cm for patients with p16+/+, p16+/-, and p16-/- biopsies, respectively (P < 0.001). We conclude that most Barrett's metaplasia contains genetic and/or epigenetic p16 lesions and has the ability to undergo clonal expansion, creating a field in which other abnormalities can arise that can lead to esophageal adenocarcinoma.

Frequent loss of heterozygosity at the retinoblastoma locus in human esophageal cancers.

Abnormalities in the retinoblastoma tumor suppressor gene (Rb) have been observed in a large number of human cancers. Loss of heterozygosity is a common mode of allelic inactivation of Rb and other tumor suppressor genes. We investigated DNA from 61 primary human esophageal tumors for loss of heterozygosity at the Rb locus using a polymerase chain reaction-based restriction fragment length polymorphism assay. Of informative cases, we found loss of heterozygosity in 14 of 26 (54%) squamous cell carcinomas and 5 of 14 (36%) adenocarcinomas. These data support the hypothesis that Rb inactivation is involved in the pathogenesis and/or progression of esophageal cancer.

Loss of heterozygosity involves multiple tumor suppressor genes in human esophageal cancers.

Loss of heterozygosity occurring on various chromosomes has been described in the majority of human tumors. The targets of frequent or consistent subchromosomal deletions are believed to be tumor suppressor genes. We examined 72 esophageal tumors (46 squamous cell carcinomas and 26 adenocarcinomas) for loss of heterozygosity at the p53, Rb, APC, MCC, and DCC loci. Inclusion of these tumor suppressor genes in the allelic deletions was directly ascertained by performing polymerase chain reaction at polymorphic sites within the genes. Loss of heterozygosity occurred in 55% of informative cases at p53, in 48% of informative cases at Rb, in 66% at APC, in 63% at MCC, and in 24% at DCC. Ninety-three % of tumors informative at all loci (fully informative) lost heterozygosity of at least one locus. A high percentage of fully informative tumors (71%) also lost heterozygosity at more than one locus. There were no significant differences among histological types in the prevalence of loss of heterozygosity at any locus. There were correlations of losses involving MCC versus DCC, Rb, and p53. These data suggest that (a) allelic deletions including these tumor suppressor genes are important in the formation and/or progression of most esophageal cancers; (b) allelic deletions involving MCC may not occur independently of deletions involving other tumor suppressor genes; and (c) the accumulation of multiple allelic deletions involving specific tumor suppressor genes may be important in most esophageal tumorigenesis or tumor evolution.

Determination of the frequency of loss of heterozygosity in esophageal adenocarcinoma by cell sorting, whole genome amplification and microsatellite polymorphisms.

It is well established that the progression to human cancer is characterized by the evolution of clones of cells with accumulated genetic abnormalities. However, technical difficulties limit the ability to study this process in some premalignant and malignant conditions. For example, the progression to esophageal adenocarcinoma in the premalignant condition Barrett's esophagus is characterized by the evolution of genetic and cell cycle abnormalities, but it has been difficult to characterize this process completely because of the small size of biopsies and the relative abundance of genetically normal stromal cells in some esophageal adenocarcinomas and premalignant mucosa. We have combined flow cytometric cell sorting to obtain purified populations of neoplastic cells with whole genome amplification and analysis of microsatellite polymorphisms to determine the frequency of allelic loss on every nonacrocentric autosomal arm in 20 esophageal adenocarcinomas and two high-grade dysplasias. DNA samples of purified flow-sorted aneuploid and corresponding normal tissue were amplified with a degenerate 15mer primer. Aliquots of these reactions were then screened with forty-three highly polymorphic simple sequence repeat markers in PCR-based assays. Allelic losses were observed at polymorphic loci in 38 of the 40 chromosome arms that were analysed and the median fractional allelic loss (FAL) observed in the samples was 0.28. The background allelic loss frequency was estimated at 0.23 with the highest rates of loss observed at 17p (100%), 5q (80%), 9p (64%), 13q (43%), 18q (43%) and 1p (41%). These data represent the first comprehensive allelotype of esophageal adenocarcinoma and show the feasibility of multiloci analyses with small highly purified human biopsy material.

Allelic loss of 9p21 and mutation of the CDKN2/p16 gene develop as early lesions during neoplastic progression in Barrett's esophagus.

High frequency allelic loss of chromosome 9p21 has been reported in a number of human cancers, including those of the esophagus. The CDKN2 gene on chromosome 9p21 that encodes the p16 inhibitor of cyclinD/Cdk4 complexes is a target of allelic loss and inactivation in a variety of human cancers and cell lines. However, the roles of 9p21 allelic losses and CDKN2 mutations in human neoplastic progression in vivo remain controversial. We determined the prevalence of allelic loss at 9p21 and mutations in CDKN2 in esophageal adenocarcinomas and investigated the order in which they occurred relative to the development of aneuploidy and cancer during neoplastic progression. Aneuploid cell populations from 32 patients with Barrett's esophagus who had premalignant epithelium, cancer, or both, were purified by DNA content flow cytometric cell sorting and evaluated by polymerase chain reaction. Twenty-four of 32 informative patients (75%) had allelic loss at 9p21 in aneuploid cell populations. Premalignant epithelium was available for seven of the patients who had 9p21 allelic losses in cancer; allelic loss of 9p21 was detected before cancer in all seven (100%). Allelic loss of 9p21 preceded the development of aneuploidy in 13 of 15 patients (87%) who had aneuploid cell populations detected in premalignant epithelium, and the two events were detected simultaneously in the remaining two patients. Five of 22 aneuploid populations (23%) with 9p21 loss had somatic mutations in the remaining CDKN2 allele. The same mutations and 9p21 allelic losses were also found in the corresponding diploid cells from premalignant epithelium in all three cases that were evaluable. However, there was no evidence for mutation or homozygous deletion of p16 in the other 17 patients with 9p21 allelic loss. Our results indicate that 9p21 allelic losses and CDKN2 mutations develop as early lesions in diploid cells before aneuploidy and cancer during neoplastic progression in Barrett's esophagus.

Hereditary gastrointestinal polyposis syndromes.

Hereditary gastrointestinal polyposis syndromes can be divided into adenomatous and hamartomatous types. Familial adenomatous polyposis coli (FAPC) is the prototype adenomatous polyposis syndrome and is defined by the autosomal dominant transmission of multiple (more than 100) colorectal adenomas. Virtually all affected patients develop colorectal carcinoma if untreated. Adenomas may develop also in the stomach and small bowel in FAPC patients, but the incidence of carcinoma in these sites is low. A variety of extracolonic manifestations has been reported in FAPC, with the name Gardner's syndrome applied to kindreds with osteomas of the skull and mandible, multiple epidermal cysts, and other skin and soft-tissue lesions. In Turcot's syndrome, brain tumors are present. The distinction between Gardner's and Turcot's syndromes and classical FAPC has become blurred because of marked overlap between them; some authorities consider them to be varying manifestations of a single genetic defect. The hamartomatous polyposes include Peutz-Jeghers syndrome, familial juvenile polyposis, Cowden's disease, intestinal ganglioneuromatosis, and the Ruvalcaba-Myrhe-Smith syndrome. The incidence of gastrointestinal cancer in patients with Peutz-Jeghers syndrome and familial juvenile polyposis exceeds that in the normal population, but is relatively low. In Cowden's disease, the gastrointestinal tract may be the site of multiple hamartomas, but there is no associated increase in the incidence of gastrointestinal cancers; instead, there is an increased incidence of carcinoma of the breast and thyroid. Intestinal ganglioneuromatosis occurs in von Recklinghausen's disease, in association with multiple endocrine neoplasia, type 2b, or as an isolated abnormality. Patients with ganglioneuromatosis do not appear to have an increased risk of developing gastrointestinal cancer. Ruvalcaba-Myrhe-Smith syndrome comprises macrocephaly, mental deficiency, an unusual craniofacial appearance, hamartomatous intestinal polyposis, and pigmented macules on the penis. No increased risk of developing cancer has been identified in the few reported cases.