P. Meera Khan

The International Collaborative Group on Hereditary Non-Polyposis Colorectal Cancer (ICG-HNPCC)

The International Collaborative Group on Hereditary Non-Polyposis Colorectal Cancer (ICG-HNPCC) H. Vasen;J. Mecklin;P. Meera Khan;H. Lynch; Diseases of the Colon & Rectum

Clinical findings with implications for genetic testing in families with clustering of colorectal cancer.

BACKGROUND Germ-line mutations in DNA mismatch-repair genes (MSH2, MLH1, PMS1, PMS2, and MSH6) cause susceptibility to hereditary nonpolyposis colorectal cancer. We assessed the prevalence of MSH2 and MLH1 mutations in families suspected of having hereditary nonpolyposis colorectal cancer and evaluated whether clinical findings can predict the outcome of genetic testing. METHODS We used denaturing gradient gel electrophoresis to identify MSH2 and MLH1 mutations in 184 kindreds with familial clustering of colorectal cancer or other cancers associated with hereditary nonpolyposis colorectal cancer. Information on the site of cancer, the age at diagnosis, and the number of affected family members was obtained from all families. RESULTS Mutations of MSH2 or MLH1 were found in 47 of the 184 kindreds (26 percent). Clinical factors associated with these mutations were early age at diagnosis of colorectal cancer, the occurrence in the kindred of endometrial cancer or tumors of the small intestine, a higher number of family members with colorectal or endometrial cancer, the presence of multiple colorectal cancers or both colorectal and endometrial cancers in a single family member, and fulfillment of the Amsterdam criteria for the diagnosis of hereditary nonpolyposis colorectal cancer (at least three family members in two or more successive generations must have colorectal cancer, one of whom is a first-degree relative of the other two; cancer must be diagnosed before the age of 50 in at least one family member; and familial adenomatous polyposis must be ruled out). Multivariate analysis showed that a younger age at diagnosis of colorectal cancer, fulfillment of the Amsterdam criteria, and the presence of endometrial cancer in the kindred were independent predictors of germ-line mutations of MSH2 or MLH1. These results were used to devise a logistic model for estimating the likelihood of a mutation in MSH2 and MLH1. CONCLUSIONS Assessment of clinical findings can improve the rate of detection of mutations of DNA mismatch-repair genes in families suspected of having hereditary nonpolyposis colorectal cancer.

Enzyme electrophoresis on cellulose acetate gel: Zymogram patterns in man-mouse and man-Chinese hamster somatic cell hybrids

Because of the occurrence of preferential loss of human chromosomes and the existance of built-in enzyme markers, the man-mouse and man-Chinese hamster somatic cell hybrid systems have become important tools for genetic analysis of man. Cellulose acetate gel (Celloged) was found to have certain additional advantages over the other supporting media for zymogram analysis. Therefore, techniques, developed during the course of a series of investigations on somatic cell hybrids in our laboratory, are described for the electrophoretic characterization of glycose-6-phosphate dehydrogenase, nicotinamide adenine dinucleotide phosphate-dependant isocitrate dehydrogenase, lactate dehydrogenase, nicotinamide-adenine dinucleotide-dependant malate dehydrogenase, 6-phosphoglyconate dehydrogenase, indophenode oxidase (1), hypoxanthine-guanine phosphoribosyl transferase, phosphoglucomutase, and phosphoglycerate kinase 2 in the man-mouse and man-Chinese hamster somatic cell hybrid systems, on Cellogel. The results are briefly discussed.

Hereditary nonpolyposis colorectal cancer families not complying with the Amsterdam criteria show extremely low frequency of mismatch-repair-gene mutations.

Hereditary nonpolyposis colorectal cancer (HNPCC) is a common autosomal dominant cancer-susceptibility condition characterized by early onset colorectal cancer. Germ-line mutations in one of four DNA mismatch repair (MMR) genes, hMSH2, hMLH1, hPMS1, or hPMS2, are known to cause HNPCC. Although many mutations in these genes have been found in HNPCC kindreds complying with the so-called Amsterdam criteria, little is known about the involvement of these genes in families not satisfying these criteria but showing clear-cut familial clustering of colorectal cancer and other cancers. Here, we applied denaturing gradient-gel electrophoresis to screen for hMSH2 and hMLH1 mutations in two sets of HNPCC families, one set comprising families strictly complying with the Amsterdam criteria and another set in which at least one of the criteria was not satisfied. Interestingly, hMSH2 and hMLH1 mutations were found in 49% of the kindreds fully complying with the Amsterdam criteria, whereas a disease-causing mutation could be identified in only 8% of the families in which the criteria were not satisfied fully. In correspondence with these findings, 4 of 6 colorectal tumors from patients belonging to kindreds meeting the criteria showed microsatellite instability, whereas only 3 of 11 tumors from the other set of families demonstrated this instability. Although the number of tumors included in the study admittedly is small, the frequencies of mutations in the MMR genes show obvious differences between the two clinical sets of families. These results also emphasize the practical importance of the Amsterdam criteria, which provide a valid clinical subdivision between families, on the basis of their chance of carrying an hMSH2 or an hMLH1 mutation, and which bear important consequences for genetic testing and counseling and for the management of colorectal cancer families.

A cost-effectiveness analysis of colorectal screening of hereditary nonpolyposis colorectal carcinoma gene carriers

It has been estimated that the prevalence of carriers of a mutated mismatch repair (MMR) gene among the general population in Western countries is between 5 and 50 per 10,000. These carriers have a risk of >85% of developing colorectal carcinoma (CRC) and therefore need careful follow‐up. The objective of this study was to analyze the cost‐effectiveness of CRC surveillance for carriers of a mutated MMR gene.

At least four different chromosomal regions are involved in loss of heterozygosity in human breast carcinoma

Three chromosome regions, i.e., 11p15, 13q, and 17p, were previously reported by three independent groups to be specifically reduced to hemizygosity in human primary breast cancer. We examined the DNA of 64 mammary tumors for loss of heterozygosity (LOH) with 28 polymorphic DNA markers dispersed on 10 arms of 8 different chromosomes. Complete or near-complete absence of LOH was observed on 5 arms (5 chromosomes). LOH at all three previously invoked regions was confirmed, and the highest frequency was found on 17p (67% of heterozygous patients). Allele loss of a marker from chromosome 3 (region p14-p21) was found in 7 of 15 informative cases. Concurrent LOH at 2 to 4 loci was noted in 20 of the 43 tumors showing LOH. Allele losses did not correlate with any of the six clinico-histopathological variables investigated, but in a group of patients in which we were unable to demonstrate LOH, the absence of distant metastases was statistically significant (P less than 0.05). These results suggest that some of the observed allele losses reflect random events, possibly as a result of genetic instability, but are not without biological significance for the progression of particular subclasses of breast tumors.

Molecular genetic tests as a guide to surgical management of familial adenomatous polyposis

BACKGROUND In familial adenomatous polyposis the only curative treatment is colectomy, and the choice of operation lies between restorative proctocolectomy (RPC) and colectomy with ileorectal anastomosis (IRA). The RPC procedure carries a higher morbidity but, unlike IRA, removes the risk of subsequent rectal cancer. Since the course of familial adenomatous polyposis is influenced by the site of mutation in the polyposis gene, DNA analysis might be helpful in treatment decisions. METHODS We evaluated the incidence of rectal cancer in polyposis patients who had undergone IRA, and examined whether the requirement for subsequent rectal excision because of cancer or uncontrollable polyps was related to the site of mutation. FINDINGS Between 1956 and mid-1995, 225 patients registered at the Netherlands Polyposis Registry had undergone IRA. In 87 of them, a pathogenetic mutation was detected. 72 patients had a mutation located before codon 1250 and 15 patients after this codon. The cumulative risk of rectal cancer 20 years after surgery was 12%, and at that time 42% had undergone rectal excision. The risk of secondary surgery was higher in patients with mutations in the region after codon 1250 than in patients with mutations before this codon (relative risk 2.7, p < 0.05). INTERPRETATION On this evidence, IRA should be the primary treatment for polyposis in patients with mutations before codon 1250, and RPC in those with mutations after this codon.

Germline mutations in the 3′ part of APC exon 15 do not result in truncated proteins and are associated with attenuated adenomatous polyposis coli

Abstract Familial adenomatous polyposis (FAP) is an inherited predisposition to colorectal cancer characterized by the development of numerous adenomatous polyps predominantly in the colorectal region. Germline mutations in the adenomatous polyposis coli (APC) gene are responsible for most cases of FAP. Mutations at the 5′ end of APC are known to be associated with a relatively mild form of the disease, called attenuated adenomatous polyposis coli (AAPC). We identified a frameshift mutation in the 3′ part of exon 15, resulting in a stop codon at 1862, in a large Dutch kindred with AAPC. Western blot analysis of lymphoblastoid cell lines derived from affected family members from this kindred, as well as from a previously reported Swiss family carrying a frameshift mutation at codon 1987 and displaying a similar attenuated phenotype, showed only the wild-type APC protein. Our study indicates that chain-terminating mutations located in the 3′ part of APC do not result in detectable truncated polypeptides and we hypothesize that this is likely to be the basis for the observed AAPC phenotype.

Molecular analysis of the APC gene in 105 Dutch kindreds with familial adenomatous polyposis: 67 germline mutations identified by DGGE, PTT, and southern analysis.

Germline mutations of the adenomatous polyposis coli (APC) gene are responsible for familial adenomatous polyposis (FAP), an autosomal dominant predisposition to colorectal cancer. We screened the entire coding region of the APC gene for mutations in an unselected series of 105 Dutch FAP kindreds. For the analysis of exons 1–14, we employed the GC‐clamped denaturing gradient gel electrophoresis (DGGE), while the large exon 15 was examined using the protein truncation test. Using this approach, we identified 65 pathogenic mutations in the above 105 apparently unrelated FAP families. The mutations were predominantly either frameshifts (39/65) or single base substitutions (18/65), resulting in premature stop codons. Mutations that would predict abnormal RNA splicing were identified in seven cases. In one of the families, a nonconservative amino acid change was found to segregate with the disease. In spite of the large number of APC mutations reported to date, we identified 27 novel germline mutations in our patients, which reiterates the great heterogeneity of the mutation spectrum in FAP. In addition to the point mutations identified in our patients, structural rearrangements of APC were found in two pedigrees, by Southern blot analysis. The present study indicates that the combined use of DGGE, protein truncation test, and Southern blot analysis offers an efficient strategy for the presymptomatic diagnosis of FAP by direct mutation detection. We found that the combined use of the currently available molecular approaches still fails to identify the underlying genetic defect in a significant subset of the FAP families. The possible causes for this limitation are discussed.

Apc1638N: A mouse model for familial adenomatous polyposis–associated desmoid tumors and cutaneous cysts☆☆☆

BACKGROUND & AIMS Germline mutations in the adenomatous polyposis coli (APC) gene are responsible for familial adenomatous polyposis (FAP), an autosomal dominant predisposition to the formation of multiple colorectal adenomas. Moreover, patients with FAP are at high risk of developing several extracolonic manifestations, including desmoids, cutaneous cysts, and tumors of the upper gastrointestinal tract. Although by definition desmoids are nonmalignant, because of their aggressive invasion of local structures, they represent one of the major causes of morbidity and mortality among patients with FAP. METHODS This study describes the histopathologic and molecular characterization of Apc1638N, a mouse model for the broad spectrum of extracolonic manifestations characteristic of FAP. RESULTS Heterozygous Apc+/Apc1638N animals develop fully penetrant and multifocal cutaneous follicular cysts and desmoid tumors in addition to attenuated polyposis of the upper gastrointestinal tract. Moreover, breeding of Apc+/Apc1638N mice in a p53-deficient background results in a dramatic seven-fold increase of the desmoid multiplicity. CONCLUSIONS Because of the attenuated nature of their intestinal phenotype, these mice survive longer than other murine models for Apc-driven tumorigenesis. Therefore, Apc1638N represents an ideal laboratory tool to test various therapeutic intervention strategies for the management of intestinal as well as extraintestinal tumors.