MSH2 deficient mice are viable and susceptible to lymphoid tumours.

Alterations of the human MSH2 gene, a homologue of the bacterial MutS mismatch repair gene, co-segregate with the majority of hereditary non-polyposis colon cancer (HNPCC) cases. We have generated homozygous MSH2-/- mice. Surprisingly, these mice were found to be viable, produced offspring in a mendelian ratio and bred through at least two generations. Starting at two months of age homozygous-/- mice began, with high frequency, to develop lymphoid tumours that contained microsatellite instabilities. These data establish a direct link between MSH2 deficiency and the pathogenesis of cancer. These mutant mice should be good models to study the progression of tumours and also to screen carcinogenic and anti-cancer agents.

Complications of the lateral transpsoas approach for lumbar interbody arthrodesis: a case series and literature review.

BACKGROUND: The lateral transpsoas approach to the lumbar spine was developed to eliminate the need for an anterior-approach surgeon and retraction of the great vessels and has the potential for shorter operative times. However, the reported complications associated with this approach vary. QUESTIONS/PURPOSES: We identified the incidence of complications associated with the lateral transpsoas approach to the lumbar spine. PATIENTS AND METHODS: We retrospectively reviewed 45 patients who underwent a lateral transpsoas approach to the spine for various diagnoses between January 1, 2006, and October 31, 2010. The patients' average age was 63.3 years. Sixteen (35.6%) patients had prior lumbar spinal surgery. Twenty-one patients (46.7%) underwent supplemental posterior instrumentation. Minimum followup was 0 months (mean, 11 months; range, 0-34 months). RESULTS: Eighteen of the 45 patients (40%) had complications: 10 (22.2%) developed postoperative iliopsoas weakness, three had quadriceps weakness, and one experienced foot drop. Eight patients (17.8%) developed anterior thigh hypoesthesia, which did not fully resolve in seven of the eight patients at an average of 9 months' followup. Three patients had postoperative radiculopathies, one a durotomy, and one died postoperatively from a pulmonary embolism. CONCLUSIONS: We found a 40% incidence of complications and a nontrivial frequency and severity of postoperative weakness, numbness, and radicular pain in patients who underwent a lateral transpsoas approach to the spine. Given the expanding use of the approach, a thorough understanding of the risks associated with it is essential for patient education, medical decision making, and identifying methods of reducing such complications. LEVEL OF EVIDENCE: Level IV, therapeutic study. See Guidelines for Authors for a complete description of levels of evidence.

Somatic APC and K-ras codon 12 mutations in aberrant crypt foci from human colons.

Aberrant crypt foci (ACF) are microscopic lesions which have been postulated to precede the development of adenomatous polyps, the precursors to colorectal cancer. APC and ras gene mutations have been shown to be important early molecular events in the development of colorectal neoplasms. The objective of this study was to establish the nature and frequency of these two genetic alterations in ACF harvested from human colorectal resection specimens. One hundred and fifty-four ACF comprised of between 1 and 56 crypts were harvested from the grossly normal mucosa of colorectal resection specimens of 28 patients with varying pathological diagnoses. One hundred and twenty-five ACF from 20 colons were screened for the presence of K-ras codon 12 mutations with a polymerase chain reaction/restriction enzyme-based method. The APC gene mutation cluster region was screened in 65 ACF from 20 colons using a polymerase chain reaction/single strand conformation polymorphism technique. Putative mutations were confirmed by direct sequencing. K-ras codon 12 mutations were identified in 13% (16 of 125) of ACF. We also identified APC mutations in 4.6% (3 of 65) of ACF. The results of this study demonstrate that both APC and K-ras mutations occur in ACF. These observations support the role of the ACF as a colorectal cancer precursor and provide further insight into the early genetic changes which occur during colorectal tumorigenesis.

A truncated hMSH2 transcript occurs as a common variant in the population: implications for genetic diagnosis.

Germline mutations of the hMSH2 gene are responsible for many cases of hereditary nonpolyposis colorectal cancer. While screening for hMSH2 gene mutations in hereditary nonpolyposis colorectal cancer kindreds, we observed that a previously reported germline mutation is in fact a common, alternatively spliced variant in the population. Using RT-PCR and the protein truncation test, the hMSH2 exon 13 deletion variant was found in more than 90% of individuals. The exon 13 deletion transcript was only present in lymphocyte RNA, no abnormalities were detected in genomic DNA flanking exon 13, and the deletion transcript is apparently not translated. These findings highlight further that caution should be exercised in providing genetic risk assessment on the basis of currently used germline mutation detection strategies.

MSH2 deficiency contributes to accelerated APC-mediated intestinal tumorigenesis.

Accelerated intestinal tumorigenesis is probable in hereditary nonpolyposis colorectal cancer, a condition associated with germ line DNA mismatch repair (MMR) gene defects, and is believed to be caused by rapid accumulation of replication errors in critical genes, such as the APC (adenomatous polyposis coli) tumor suppressor gene. To study the potential contribution of MMR genes to accelerated intestinal tumorigenesis, we crossed the Min mouse, heterozygous for a germ line mutation of Apc, with an MMR gene (Msh2)-deficient mouse. MSH2 deficiency resulted in the development of many colonic aberrant crypt foci, as well as reduced survival of the mice, secondary to both a greater number and more rapidly developing adenomas. The mechanism of inactivation of the wild-type Apc allele depended on MSH2 status. In the presence of functional MSH2, all tumors demonstrated loss of heterozygosity. In contrast, whereas all adenomas were APC negative by immunostaining, only 5 of 34 adenomas from Apc+/-/Msh2-/- mice demonstrated loss of heterozygosity of the wild-type Apc allele, suggesting that somatic Apc mutations are responsible for the additional tumors. These findings provide evidence for the important role of MMR genes in accelerated intestinal tumorigenesis, thus supporting more aggressive surveillance strategies to prevent colorectal cancer in hereditary nonpolyposis colorectal cancer.

Somatic APC and K-ras codon 12 mutations in periampullary adenomas and carcinomas from familial adenomatous polyposis patients.

Periampullary adenomas in the duodenum of Familial Adenomatous Polyposis (FAP) patients are among the most frequent and clinically important extracolonic neoplasms in FAP. The purpose of this study was to characterize the frequency and nature of somatic adenomatous polyposis coli (APC) gene and K-ras codon 12 mutations in periampullary adenomas and carcinomas in FAP. These molecular changes have been shown to be important during the early stages of colorectal carcinogenesis. DNA was prepared from endoscopic periampullary biopsies and paraffin blocks from 49 FAP patients. Of 143 samples, 77 were histologically normal, 29 were biopsies from small periampullary adenomas, 29 biopsies were from 19 large adenomas and eight samples were from periampullary cancers. APC mutations in the mutation cluster region and K-ras codon 12 mutations were detected by polymerase chain reaction based techniques. Somatic APC mutations consisting of deletions at codons 1464 and 1465 were detected in one small and two large periampullary adenomas. Loss of heterozygosity was seen in one periampullary carcinoma. K-ras codon 12 mutations were detected in seven of 19 large periampullary adenomas and in one of eight periampullary carcinomas. These data suggest that K-ras codon 12 mutations may be important during periampullary tumorigenesis in FAP but somatic APC mutations in the mutation cluster region are infrequent. Local environmental factors in the duodenum may contribute to differences in the molecular changes which occur during the adenoma-to-carcinoma sequence in periampullary compared to colonic tumorigenesis.

Improved predictive carrier testing for familial adenomatous polyposis using DNA from a single archival specimen and polymorphic markers with multiple alleles.

Familial adenomatous polyposis (FAP) is an autosomal dominant inherited condition caused by mutations in a candidate tumor suppressor gene, adenomatous polyposis coli. Predictive carrier testing for FAP can be accomplished by DNA linkage or mutation detection analysis. In many FAP families all affected individuals are deceased. Archival tissue specimens, if available from such patients, are a useful source of DNA for molecular analysis. We investigated the carrier risk of a presymptomatic 5-year-old girl with a family history of FAP. An archival specimen of normal colonic tissue was only available from the proband's affected deceased father. We screened DNA extracted from this specimen with dinucleotide repeat (CA/GT)n polymorphic marker (D5S346) linked to the adenomatous polyposis coli gene and established that the proband is at more than 99% risk of developing FAP. The proband's increased risk status was subsequently confirmed by identification of a germline adenomatous polyposis coli gene mutation consisting of a 5 base pair deletion at codon 1061. This strategy of using DNA from archival specimens of affected, unavailable FAP patients will increase the number of at-risk individuals that can be diagnosed presymptomatically.

Characterization of two novel adenomatous polyposis coli (APC) gene mutations in patients with familial adenomatous polyposis (FAP).

Patients with Familial Adenomatous Polyposis (FAP) manifest numerous colorectal adenomas as well as benign and malignant extra-colonic lesions. Adenomatous polyposis coli (APC) gene mutations are the underlying genetic defect in FAP. We analyzed germline DNA of 81 unrelated FAP patients and evaluated correlation of APC mutation genotype and clinical phenotype. Germline APC mutations were identified in 18 FAP patients including two novel 2 bp deletions at APC codons 1067 and 1259. FAP patients were screened for hypertrophic ocular fundus lesions, desmoids and peri-ampullary adenomas. As reported previously (Olshwang et al 1993b), a positive correlation for the frequency of retinal lesions and germline APC mutation was observed among all FAP patients except one. No significant correlation was observed for APC mutation genotype and the occurrence of desmoids and peri-ampullary adenomas. Genetic factors contributing to familial segregation of these lesions need further investigation.

Genotype-phenotype correlations in attenuated adenomatous polyposis coli.

Germ-line mutations of the tumor suppressor APC are implicated in attenuated adenomatous polyposis coli (AAPC), a variant of familial adenomatous polyposis (FAP). AAPC is recognized by the occurrence of <100 colonic adenomas and a later onset of colorectal cancer (age >40 years). The aim of this study was to assess genotype-phenotype correlations in AAPC families. By protein-truncation test (PTT) assay, the entire coding region of the APC gene was screened in affected individuals from 11 AAPC kindreds, and their phenotypic differences were examined. Five novel germ-line APC mutations were identified in seven kindreds. Mutations were located in three different regions of the APC gene: (1) at the 5' end spanning exons 4 and 5, (2) within exon 9, and (3) at the 3' distal end of the gene. Variability in the number of colorectal adenomas was most apparent in individuals with mutations in region 1, and upper-gastrointestinal manifestations were more severe in them. In individuals with mutations in either region 2 or region 3, the average number of adenomas tended to be lower than those in individuals with mutations in region 1, although age at diagnosis was similar. In all AAPC kindreds, a predominance of right-sided colorectal adenomas and rectal polyp sparing was observed. No desmoid tumors were found in these kindreds. Our data suggest that, in AAPC families, the location of the APC mutation may partially predict specific phenotypic expression. This should help in the design of tailored clinical-management protocols in this subset of FAP patients.

A germline mutation at the extreme 3' end of the APC gene results in a severe desmoid phenotype and is associated with overexpression of beta-catenin in the desmoid tumor.

Desmoid tumors arise sporadically or as part of the extraintestinal manifestations of familial adenomatous polyposis (FAP). In FAP, two distinct clinical presentations of the desmoid phenotype are seen: 1) one or a few desmoid tumors present predominantly in the abdominal wall or the abdomen; 2) a florid proliferation of tumors early in life, mostly near the axial skeleton or extremities. These different phenotypes have been associated with different sites of germline mutations in the adenomatous polyposis coli gene (APC gene). We present a large, French-Canadian kindred with a florid desmoid tumor phenotype caused by a germline mutation at codon 2643-2644 of the APC gene. The phenotype was characterized by the early onset of multiple tumors, arising near the axial skeleton and in proximal extremities. The penetrance of desmoid tumors was near 100% in this kindred. However, the expression of the disease was variable amongst the different affected relatives. Many gene carriers had cutaneous cysts. Polyposis of the colon was rarely observed in the affected individuals and we did not document upper gastro-intestinal polyps. The mutant APC allele did not express a stable truncated protein in vivo. Molecular analysis of the proband's tumor DNA revealed a somatic inactivating mutation of the wild-type allele. Immunohistochemistry on the tumor also demonstrated elevated levels of beta-catenin. The present study demonstrates that this extreme 3' APC mutation is associated with a severely penetrant desmoid phenotype and attenuated polyposis coli. It also suggests the involvement of the beta-catenin pathway in the development of desmoid tumors in FAP. The natural history of the disease is variable between individuals, and surgical interventions have to be timed appropriately due to the frequent recurrences.

Mismatch repair gene defects contribute to the genetic basis of double primary cancers of the colorectum and endometrium.

Hereditary non-polyposis colorectal cancer (HNPCC) is a dominantly inherited cancer syndrome caused by germline defects of mismatch repair (MMR) genes. Endometrial cancer is the most common extracolonic neoplasm in HNPCC and is the primary clinical manifestation of the syndrome in some families. The cumulative incidence of endometrial cancer among HNPCC mutation carriers is high, estimated to be from 22 to 43%. We hypothesized that women with double primary cancers of the colorectum and endometrium are likely to be members of HNPCC families. In order to determine how frequently HNPCC manifests in the context of double primary cancers, we examined alterations of two MMR genes, hMSH2 and hMLH1, in 40 unrelated women affected with double primary cancers. These cases were identified using hospital-based and population-based cancer registries in Ontario, Canada. MMR gene mutations were screened by single-strand conformation polymorphism analysis and confirmed by direct sequencing. Eighteen percent (seven of 40) were found to harbor mutations of one of the two MMR genes. Analysis of colorectal and/or endometrial tumors of mutation-negative probands found microsatellite instability in seven of 20 cases. Six of seven mutation-positive probands had strong family histories suggestive of HNPCC. First degree relatives of mutation-positive probands had a very high relative risk (RR) of colorectal cancer (RR = 8.1, CI 3. 5-15.9) and endometrial cancer (RR = 23.8, CI 6.4-61.0). The relative risk of mutation-negative cases was 2.8 (CI 1.7-4.5) for colorectal cancer and 5.4 (CI 2.0-11.7) for endometrial cancer. We recommend that all double primary patients with cancers at these sites should have a genetic evaluation, including molecular analysis for HNPCC where appropriate.

Cost comparison of predictive genetic testing versus conventional clinical screening for familial adenomatous polyposis.

BACKGROUND: Mutations of the APC gene cause familial adenomatous polyposis (FAP), a hereditary colorectal cancer predisposition syndrome. AIMS: To conduct a cost comparison analysis of predictive genetic testing versus conventional clinical screening for individuals at risk of inheriting FAP, using the perspective of a third party payer. METHODS: All direct health care costs for both screening strategies were measured according to time and motion, and the expected costs evaluated using a decision analysis model. RESULTS: The baseline analysis predicted that screening a prototype FAP family would cost $4975/ pound3109 by molecular testing and $8031/ pound5019 by clinical screening strategy, when family members were monitored with the same frequency of clinical surveillance (every two to three years). Sensitivity analyses revealed that the genetic testing approach is cost saving for key variables including the kindred size, the age of screening onset, and the cost of mutation identification in a proband. However, if the APC mutation carriers were monitored at an increased (annual) frequency, the cost of the genetic screening strategy increased to $7483/ pound4677 and was especially sensitive to variability in age of onset of screening, family size, and cost of genetic testing of at risk relatives. CONCLUSIONS: In FAP kindreds, a predictive genetic testing strategy costs less than conventional clinical screening, provided that the frequency of surveillance is identical using either strategy. An additional significant benefit is the elimination of unnecessary colonic examinations for those family members found to be non-carriers.

Familial adenomatous polyposis-associated thyroid cancer: a clinical, pathological, and molecular genetics study.

We report two familial adenomatous polyposis (FAP) kindreds with thyroid cancer, harboring two apparently novel germlineAPC mutations. The clinical phenotype in the first kindred was typical of classical adenomatous polyposis, whereas the second kindred exhibited an attenuated adenomatous polyposis phenotype. There was a female predominance with a mean age of 34 years (range, 23-49) at cancer diagnosis. Multiple sections of four thyroid tumors from three FAP patients were analyzed in detail. Histological examination of thyroid tumors showed a range of morphological features. Some tumors exhibited typical papillary architecture and were associated with multifocal carcinoma; in others, there were unusual areas of cribriform morphology, and spindle-cell components with whorled architecture. Immunoreactivity for thyroglobulin and high molecular weight keratins was strong. Somatic APC mutation analysis revealed an insertion of a novel long interspersed nuclear element-1-like sequence in one tumor sample, suggesting disruption of APC. In three FAP patients, ret/PTC-1 and ret/PTC-3 were expressed in thyroid cancers. No positivity was observed for ret/ PTC-2. p53 immunohistochemistry was positive in only one section of a recurrent thyroid tumor sample. Our data suggest that genetic alterations in FAP-associated thyroid cancer involve loss of function of APC along with the gain of function of ret/PTC, while alterations of p53 do not appear to be an early event in thyroid tumorigenesis.

Family history characteristics, tumor microsatellite instability and germline MSH2 and MLH1 mutations in hereditary colorectal cancer.

Recent characterization of the molecular genetic basis of hereditary nonpolyposis colorectal cancer provides an important opportunity for identification of individuals and their families with germline mutations in mismatch repair genes. Cancer family history criteria that accurately define hereditary colorectal cancer are necessary for cost-effective testing for germline mutations in mismatch repair genes. The present report describes the results of analysis of 33 colorectal cancer cases/families that satisfy our modified family history criteria (Mount Sinai criteria) for colorectal cancer. Fourteen of these families met the more stringent Amsterdam criteria. Germline MSH2 and MLH1 mutations were identified by the reverse transcription-polymerase chain reaction and the protein truncation test, and confirmed by sequencing. Microsatellite instability analysis was performed on available tumors from affected patients. MSH2 or MLH1 mutations were detected in 8 of 14 Amsterdam criteria families and in 5 of the remaining 19 cases/families that only satisfied the Mount Sinai criteria. Three of the latter families had features of the Muir-Torre syndrome. A high level of microsatellite instability (MSI-H) was detected in almost all (16/18) colorectal cancers from individuals with MSH2 and MLH1 mutations, and infrequently (1/21) in colorectal cancer specimens from cases without detectable mutations. Families with germline MSH2 and MLH1 mutations tended to have individuals affected at younger ages and with multiple tumors. The Amsterdam criteria are useful, but not sufficient, for detecting hereditary colorectal cancer families with germline MSH2 and MLH1 mutations, since a proportion of cases and families with mutations in mismatch repair genes will be missed. Further development of cancer family history criteria are needed, using unbiased prospectively collected cases, to define more accurately those who will benefit from MSH2 and MLH1 mutation analysis.