Bharati Bapat

MADR2 Maps to 18q21 and Encodes a TGFβ–Regulated MAD–Related Protein That Is Functionally Mutated in Colorectal Carcinoma

The MAD-related (MADR) family of proteins are essential components in the signaling pathways of serine/threonine kinase receptors for the transforming growth factor beta (TGFbeta) superfamily. We demonstrate that MADR2 is specifically regulated by TGFbeta and not bone morphogenetic proteins. The gene for MADR2 was found to reside on chromosome 18q21, near DPC4, another MADR protein implicated in pancreatic cancer. Mutational analysis of MADR2 in sporadic tumors identified four missense mutations in colorectal carcinomas, two of which display a loss of heterozygosity. Biochemical and functional analysis of three of these demonstrates that the mutations are inactivating. These findings suggest that MADR2 is a tumor suppressor and that mutations acquired in colorectal carcinomas may function to disrupt TGFbeta signaling.

Predominance of beta-catenin mutations and beta-catenin dysregulation in sporadic aggressive fibromatosis (desmoid tumor).

Aggressive fibromatosis (also called desmoid tumor) occurs as a sporadic lesion or as part of Familial Adenomatous Polyposis, which is caused by germ line mutations in the Adenomatous polyposis Coli (APC) gene. APC is involved in the regulation of the cellular level of beta-catenin, which is a mediator in Wnt signaling. Mutational analysis of the beta-catenin and APC genes was performed in 42 sporadic aggressive fibromatoses. Nine tumors had mutations in APC, and 22 had a point mutation in beta-catenin at either codon 45 or codon 41 (producing a stabilized beta-catenin protein product). Immunohistochemistry showed an elevated beta-catenin protein level in all tumors, regardless of mutational status. Beta-catenin localized to the nucleus, and was not tyrosine phosphorylated in the six tumors in which this was tested. The demonstration of mutations in two mediators in the Wnt-APC-beta-catenin pathway implicates beta-catenin stabilization as the key factor in the pathogenesis of aggressive fibromatosis. This is the first demonstration of somatic beta-catenin mutations in a locally invasive, but non metastatic lesion composed of spindle cells, illustrating the importance of beta-catenin stabilization in a variety of cell types and neoplastic processes. Moreover, this tumor has one of the highest reported frequencies of beta-catenin mutations of any tumor type.

Human microphthalmia associated with mutations in the retinal homeobox gene CHX10.

Isolated human microphthalmia/anophthalmia, a cause of congenital blindness, is a clinically and genetically heterogeneous developmental disorder characterized by a small eye and other ocular abnormalities. Three microphthalmia/anophthalmia loci have been identified, and two others have been inferred by the co-segregation of translocations with the phenotype. We previously found that mice with ocular retardation (the or-J allele), a microphthalmia phenotype, have a null mutation in the retinal homeobox gene Chx10 (refs 7,8). We report here the mapping of a human microphthalmia locus on chromosome 14q24.3, the cloning of CHX10 at this locus and the identification of recessive CHX10 mutations in two families with non-syndromic microphthalmia (MIM 251600), cataracts and severe abnormalities of the iris. In affected individuals, a highly conserved arginine residue in the DNA-recognition helix of the homeodomain is replaced by glutamine or proline (R200Q and R200P, respectively). Identification of the CHX10 consensus DNA-binding sequence (TAATTAGC) allowed us to demonstrate that both mutations severely disrupt CHX10 function. Human CHX10 is expressed in progenitor cells of the developing neuroretina and in the inner nuclear layer of the mature retina. The strong conservation in vertebrates of the CHX10 sequence, pattern of expression and loss-of-function phenotypes demonstrates the evolutionary importance of the genetic network through which this gene regulates eye development.

Molecular biology of colorectal cancer

Colorectal cancer is a significant cause of morbidity and mortality in Western populations. This cancer develops as a result of the pathologic transformation of normal colonic epithelium to an adenomatous polyp and ultimately an invasive cancer. The multistep progression requires years and possibly decades and is accompanied by a number of recently characterized genetic alterations. Mutations in two classes of genes, tumor-suppressor genes and proto-oncogenes, are thought to impart a proliferative advantage to cells and contribute to development of the malignant phenotype. Inactivating mutations of both copies (alleles) of the adenomatous polyposis coli (APC) gene--a tumor-suppressor gene on chromosome 5q--mark one of the earliest events in colorectal carcinogenesis. Germline mutation of the APC gene and subsequent somatic mutation of the second APC allele cause the inherited familial adenomatous polyposis syndrome. This syndrome is characterized by the presence of hundreds to thousands of colonic adenomatous polyps. If these polyps are left untreated, colorectal cancer develops. Mutation leading to dysregulation of the K-ras protooncogene is also thought to be an early event in colon cancer formation. Conversely, loss of heterozygosity on the long arm of chromosome 18 (18q) occurs later in the sequence of development from adenoma to carcinoma, and this mutation may predict poor prognosis. Loss of the 18q region is thought to contribute to inactivation of the DCC tumor-suppressor gene. More recent evidence suggests that other tumor-suppressor genes--DPC4 and MADR2 of the transforming growth factor beta (TGF-beta) pathway--also may be inactivated by allelic loss on chromosome 18q. In addition, mutation of the tumor-suppressor gene p53 on chromosome 17p appears to be a late phenomenon in colorectal carcinogenesis. This mutation may allow the growing tumor with multiple genetic alterations to evade cell cycle arrest and apoptosis. Neoplastic progression is probably accompanied by additional, undiscovered genetic events, which are indicated by allelic loss on chromosomes 1q, 4p, 6p, 8p, 9q, and 22q in 25% to 50% of colorectal cancers. Recently, a third class of genes, DNA repair genes, has been implicated in tumorigenesis of colorectal cancer. Study findings suggest that DNA mismatch repair deficiency, due to germline mutation of the hMSH2, hMLH1, hPMS1, or hPMS2 genes, contributes to development of hereditary nonpolyposis colorectal cancer. The majority of tumors in patients with this disease and 10% to 15% of sporadic colon cancers display microsatellite instability, also know as the replication error positive (RER+) phenotype. This molecular marker of DNA mismatch repair deficiency may predict improved patient survival. Mismatch repair deficiency is thought to lead to mutation and inactivation of the genes for type II TGF-beta receptor and insulin-like growth-factor II receptor. Individuals from families at high risk for colorectal cancer (hereditary nonpolyposis colorectal cancer or familial adenomatous polyposis) should be offered genetic counseling, predictive molecular testing, and when indicated, endoscopic surveillance at appropriate intervals. Recent studies have examined colorectal carcinogenesis in the light of other genetic processes. Telomerase activity is present in almost all cancers, including colorectal cancer, but rarely in benign lesions such as adenomatous polyps or normal tissues. Furthermore, genetic alterations that allow transformed colorectal epithelial cells to escape cell cycle arrest or apoptosis also have been recognized. In addition, hypomethylation or hypermethylation of DNA sequences may alter gene expression without nucleic acid mutation.

Immunohistochemistry for hMLH1 and hMSH2: a practical test for DNA mismatch repair-deficient tumors.

Inactivation of deoxyribonucleic acid (DNA) mismatch repair genes, most commonly human mutL homologue 1 (hMLH1) or human mutS homologue 2 (hMSH2), is a recently described alternate pathway in cancer development and progression. The resulting genetic instability is characterized by widespread somatic mutations in tumor DNA, and is termed high-frequency microsatellite instability (MSI-H). Although described in a variety of tumors, mismatch repair deficiency has been studied predominantly in colorectal carcinoma. Most MSI-H colorectal carcinomas are sporadic, but some occur in patients with hereditary nonpolyposis colorectal cancer (HNPCC), and are associated with germline mutations in mismatch repair genes. Until now, the identification of MSI-H cancers has required molecular testing. To evaluate the role of immunohistochemistry as a new screening tool for mismatch repair-deficient neoplasms, the authors studied the expression of hMLH1 and hMSH2, using commercially available monoclonal antibodies, in 72 formalin-fixed, paraffin-embedded tumors that had been tested previously for microsatellite instability. They compared immunohistochemical patterns of 38 MSI-H neoplasms, including 16 cases from HNPCC patients with known germline mutations in hMLH1 or hMSH2, with 34 neoplasms that did not show microsatellite instability. Thirty-seven of 38 MSI-H neoplasms were predicted to have a mismatch repair gene defect, as demonstrated by the absence of hMLH1 and/or hMSH2 expression. This included correspondence with all 16 cases with germline mutations. All 34 microsatellite-stable cancers had intact staining with both antibodies. These findings clearly demonstrate that immunohistochemistry can discriminate accurately between MSI-H and microsatellite-stable tumors, providing a practical new technique with important clinical and research applications.

Genomic Instability and Cancer

Tumorigenesis can be viewed as an imbalance between the mechanisms of cell-cycle control and mutation rates within the genes. Genomic instability is broadly classified into microsatellite instability (MIN) associated with mutator phenotype, and chromosome instability (CIN) recognized by gross chromosomal abnormalities. Three intracellular mechanisms are involved in DNA damage repair that leads to mutator phenotype. They include the nucleotide excision repair (NER), base excision repair (BER) and mismatch repair (MMR). The CIN pathway is typically associated with the accumulation of mutations in tumor suppressor genes and oncogenes. Defects in DNA MMR and CIN pathways are responsible for a variety of hereditary cancer predisposition syndromes including hereditary non-polyposis colorectal carcinoma (HNPCC), Bloom syndrome, ataxia-telangiectasia, and Fanconi anaemia. While there are many genetic contributors to CIN and MIN, there are also epigenetic factors that have emerged to be equally damaging to cell-cycle control. Hypermethylation of tumor suppressor and DNA MMR gene promoter regions, is an epigenetic mechanism of gene silencing that contributes to tumorigenesis. Telomere shortening has been shown to increase genetic instability and tumor formation in mice, underscoring the importance of telomere length and telomerase activity in maintaining genomic integrity. Mouse models have provided important insights for discovering critical pathways in the progression to cancer, as well as to elucidate cross talk among different pathways. This review examines various molecular mechanisms of genomic instability and their relevance to cancer.

Colorectal and Other Cancer Risks for Carriers and Noncarriers From Families With a DNA Mismatch Repair Gene Mutation: A Prospective Cohort Study

PURPOSE To determine whether cancer risks for carriers and noncarriers from families with a mismatch repair (MMR) gene mutation are increased above the risks of the general population. PATIENTS AND METHODS We prospectively followed a cohort of 446 unaffected carriers of an MMR gene mutation (MLH1, n = 161; MSH2, n = 222; MSH6, n = 47; and PMS2, n = 16) and 1,029 their unaffected relatives who did not carry a mutation every 5 years at recruitment centers of the Colon Cancer Family Registry. For comparison of cancer risk with the general population, we estimated country-, age-, and sex-specific standardized incidence ratios (SIRs) of cancer for carriers and noncarriers. RESULTS Over a median follow-up of 5 years, mutation carriers had an increased risk of colorectal cancer (CRC; SIR, 20.48; 95% CI, 11.71 to 33.27; P < .001), endometrial cancer (SIR, 30.62; 95% CI, 11.24 to 66.64; P < .001), ovarian cancer (SIR, 18.81; 95% CI, 3.88 to 54.95; P < .001), renal cancer (SIR, 11.22; 95% CI, 2.31 to 32.79; P < .001), pancreatic cancer (SIR, 10.68; 95% CI, 2.68 to 47.70; P = .001), gastric cancer (SIR, 9.78; 95% CI, 1.18 to 35.30; P = .009), urinary bladder cancer (SIR, 9.51; 95% CI, 1.15 to 34.37; P = .009), and female breast cancer (SIR, 3.95; 95% CI, 1.59 to 8.13; P = .001). We found no evidence of their noncarrier relatives having an increased risk of any cancer, including CRC (SIR, 1.02; 95% CI, 0.33 to 2.39; P = .97). CONCLUSION We confirmed that carriers of an MMR gene mutation were at increased risk of a wide variety of cancers, including some cancers not previously recognized as being a result of MMR mutations, and found no evidence of an increased risk of cancer for their noncarrier relatives.

Changing causes of mortality in patients with familial adenomatous polyposis.

Widespread use of prophylactic colectomy has resulted in a reduction in the incidence of colorectal cancer in familial adenomatous polyposis (FAP) patients. A retrospective chart review of families registered at the Steve Atanas Stavro Familial Gastrointestinal Cancer Registry in Toronto was performed to determine whether the decrease in the number of patients developing colorectal cancer implies that causes of mortality in FAP patients are shifting to that of extracolonic manifestations of FAP. Information was available on 140 deaths within 158 families and among 461 individuals with FAP. When stratified by decade, from the 1930s to the 1990s, the ratio of deaths caused by extracolonic manifestations of FAP compared with deaths caused by colorectal cancer was noted to have risen. Even though most deaths in FAP patients are still from colorectal cancer, it appears that screening policies and prophylactic colectomy have resulted in a reduction in the number of FAP patients who develop colorectal cancer. Thus, in recent decades, a greater percentage of deaths in FAP patients appears to be attributable to extracolonic manifestations of the disease.

Molecular Characterization of MSI-H Colorectal Cancer by MLHI Promoter Methylation, Immunohistochemistry, and Mismatch Repair Germline Mutation Screening

Microsatellite instability (MSI) occurs in 10% to 20% of colorectal cancers (CRC) and has been attributed to both MLH1 promoter hypermethylation and germline mutation in the mismatch repair (MMR) genes. We present results from a large population- and clinic-based study of MLH1 methylation, immunohistochemistry, and MMR germline mutations that enabled us to (a) estimate the prevalence of MMR germline mutations and MLH1 methylation among MSI-H cases and help us understand if all MSI-H CRC is explained by these mechanisms and (b) estimate the associations between MLH1 methylation and sex, age, and tumor location within the colon. MLH1 methylation was measured in 1,061 population-based and 172 clinic-based cases of CRC. Overall, we observed MLH1 methylation in 60% of population-based MSI-H cases and in 13% of clinic-based MSI-H cases. Within the population-based cases with MMR mutation screening and conclusive immunohistochemistry results, we identified a molecular event in MMR in 91% of MSI-H cases: 54% had MLH1 methylation, 14% had a germline mutation in a MMR gene, and 23% had immunohistochemistry evidence for loss of a MMR protein. We observed a striking age difference, with the prevalence of a MMR germline mutation more than 4-fold lower and the prevalence of MLH1 methylation more than 4-fold higher in cases diagnosed after the age of 50 years than in cases diagnosed before that age. We also determined that female sex is an independent predictor of MLH1 methylation within the MSI-H subgroup. These results reinforce the importance of distinguishing between the underlying causes of MSI in studies of etiology and prognosis. (Cancer Epidemiol Biomarkers Prev 2008;17(11):3208–15)

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.