Jennifer Rhees

De novo constitutional MLH1 epimutations confer early-onset colorectal cancer in two new sporadic Lynch syndrome cases, with derivation of the epimutation on the paternal allele in one.

Lynch syndrome is an autosomal dominant cancer predisposition syndrome classically caused by germline mutations of the mismatch repair genes, MLH1, MSH2, MSH6 and PMS2. Constitutional epimutations of the MLH1 gene, characterized by soma‐wide methylation of a single allele of the promoter and allelic transcriptional silencing, have been identified in a subset of Lynch syndrome cases lacking a sequence mutation in MLH1. We report two individuals with no family history of colorectal cancer who developed that disease at age 18 and 20 years. In both cases, cancer had arisen because of the de novo occurrence of a constitutional MLH1 epimutation and somatic loss‐of‐heterozygosity of the functional allele in the tumors. We show for the first time that the epimutation in one case arose on the paternally inherited allele. Analysis of 13 tumors from seven individuals with constitutional MLH1 epimutations showed eight tumors had lost the second MLH1 allele, two tumors had a novel pathogenic missense mutation and three had retained heterozygosity. Only 1 of 12 tumors demonstrated the BRAF V600E mutation and 3 of 11 tumors harbored a mutation in KRAS. The finding that epimutations can originate on the paternal allele provides important new insights into the mechanism of origin of epimutations. It is clear that the second hit in MLH1 epimutation‐associated tumors typically has a genetic not epigenetic basis. Individuals with mismatch repair–deficient cancers without the BRAF V600E mutation are candidates for germline screening for sequence or methylation changes in MLH1.

Identification of frame-shift intermediate mutant cells

Frame-shift mutations at microsatellites occur as a time-dependent function of polymerase errors followed by failure of postreplicational mismatch repair. A cell-culture system was developed that allows identification of intermediate mutant cells that carry the mutation on a single DNA strand after the initial DNA polymerase errors. A plasmid was constructed that contained 13 repeats of a poly(dC-dA)⋅poly(dG-dT) oligonucleotide immediately after the translation initiation codon of the enhanced GFP (EGFP) gene, shifting the EGFP gene out of its proper reading frame. The plasmid was introduced into human mismatch repair-deficient (HCT116, hMLH1-mutated) and mismatch repair-proficient (HCT116+chr3, hMLH1 wild type) colorectal cancer cells. After frame-shift mutations occurred that restored the EGFP reading frame, EGFP-expressing cells were detected, and two distinct fluorescent populations, M1 (dim cells) and M2 (bright cells), were identified. M1 cell numbers were stable, whereas M2 cells accumulated over time. In HCT116, single M2 cells gave rise to fluorescent colonies that carried a 2-bp deletion at the (CA)13 microsatellite. Twenty-eight percent of single M1 cells, however, gave rise to colonies with a mixed fluorescence pattern that carried both (CA)13 and (CA)12 microsatellites. It is likely that M1 cells represent intermediate mutants that carry (CA)13⋅(GT)12 heteroduplexes. Although the mutation rate in HCT116 cell clones (6.2 × 10−4) was 30 times higher than in HCT116+chr3 (1.9 × 10−5), the proportion of M1 cells in culture did not significantly differ between HCT116 (5.87 × 10−3) and HCT116+chr3 (4.13 × 10−3), indicating that the generation of intermediate mutants is not affected by mismatch-repair proficiency.

Prohibitin attenuates colitis-associated tumorigenesis in mice by modulating p53 and STAT3 apoptotic responses

Although inflammatory bowel disease is associated with higher risk of colorectal cancer, the precise pathogenic mechanisms underlying this association are not completely understood. Prohibitin 1 (PHB), a protein implicated in the regulation of proliferation, apoptosis, and transcription, is decreased in intestinal inflammation. In this study, we have established a key function for PHB in mediating colitis-associated cancer. Wild-type and transgenic (Tg) mice specifically overexpressing PHB in intestinal epithelial cells were subjected to a classical two-stage protocol of colitis-associated carcinogenesis. In addition, wild-type and p53 null human cell models were used to assess PHB interaction with STAT3 and p53. Wild-type mice exhibited decreased mucosal PHB protein expression during colitis-associated carcinogenesis. Tg mice exhibited decreased susceptibility in a manner associated with increased apoptosis, p53, Bax, and Bad expression plus decreased Bcl-xL and Bcl-2 expression. PHB overexpression in wild-type but not p53 null human cells increased expression of Bax, Bad, and caspase-3 cleavage. In wild-type p53 cells, PHB overexpression decreased basal and interleukin-6-induced STAT3 activation and expression of the STAT3 responsive genes Bcl-xL and Bcl-2. PHB coimmunoprecipitated with phospho-STAT3 in addition to p53 in cultured cell lysates and colon mucosa. This is the first study to show interaction between PHB and STAT3 in vivo. In summary, our findings suggest that PHB protects against colitis-associated cancer by modulating p53- and STAT3-mediated apoptosis. Modulation of PHB expression in intestinal epithelial cells may offer a potential therapeutic approach to prevent colitis-associated carcinogenesis.

1030 Somatic Hypermethylation of MSH2 is a Frequent Event in Lynch Syndrome Colorectal Cancers

Heritable germline epimutations in MSH2 have been reported in a few Lynch syndrome families that lacked germline mutations in the MSH2 gene. It is not known whether somatic MSH2 methylation occurs in MSH2 mutation–positive Lynch syndrome subjects or sporadic colorectal cancers (CRC). Therefore, we determined the methylation status of the MSH2 gene in 268 CRC tissues, including 222 sporadic CRCs and 46 Lynch syndrome tumors that did not express MSH2. We also looked for microsatellite instability (MSI), germline mutations in the MSH2 and EpCAM genes, somatic mutations in BRAF and KRAS, and the CpG island methylator phenotype (CIMP). We observed that somatic MSH2 hypermethylation was present in 24% (11 of 46) of MSH2-deficient (presumed Lynch syndrome) tumors, whereas no evidence for MSH2 methylation existed in sporadic CRCs (MSI and microsatellite stable) or normal colonic tissues. Seven of 11 (63%) patients with MSH2 methylation harbored simultaneous pathogenic germline mutations in the MSH2 gene. Germline EpCAM deletions were present in three of four patients with MSH2 methylation but without pathogenic MSH2 germline mutations. The mean methylation scores at CIMP-related markers were significantly higher in Lynch syndrome tumors with MSH2 methylation than MSH2-unmethylated CRCs. In conclusion, our data provide evidence for frequent MSH2 hypermethylation in Lynch syndrome tumors with MSH2 deficiency. MSH2 methylation in this subset of individuals is somatic and may serve as the “second hit” at the wild-type allele. High levels of aberrant methylation at CIMP-related markers in MSH2-methylated tumors raise the possibility that MSH2 is a target susceptible to aberrant methylation in Lynch syndrome. Cancer Res; 70(8); 3098–108. ©2010 AACR.

Pitfalls in the diagnosis of biallelic PMS2 mutations

Constitutional Mismatch Repair Deficiency (CMMR-D) syndrome is an inherited childhood cancer syndrome due to bi-allelic mutations in one of the four DNA mismatch repair genes involved in Lynch syndrome. The tumor spectrum of this syndrome includes hematological, brain and Lynch syndrome associated malignancies, with an increased risk of synchronous and metachronous cancers, and signs of Neurofibromatosis type-1 syndrome such as café-au-lait macules during the first three decades of life. Here, we report the first Argentinian patient with CMMR-D syndrome, focusing on her history of cancer and gastrointestinal manifestations, and the challenging molecular algorithm to finally reach her diagnosis.

Novel Mutations in MLH1 and MSH2 Genes in Mexican Patients with Lynch Syndrome

Background. Lynch Syndrome (LS) is characterized by germline mutations in the DNA mismatch repair (MMR) genes MLH1, MSH2, MSH6, and PMS2. This syndrome is inherited in an autosomal dominant pattern and is characterized by early onset colorectal cancer (CRC) and extracolonic tumors. The aim of this study was to identify mutations in MMR genes in three Mexican patients with LS. Methods. Immunohistochemical analysis was performed as a prescreening method to identify absent protein expression. PCR, Denaturing High Performance Liquid Chromatography (dHPLC), and Sanger sequencing complemented the analysis. Results. Two samples showed the absence of nuclear staining for MLH1 and one sample showed loss of nuclear staining for MSH2. The mutations found in MLH1 gene were c.2103+1G>C in intron 18 and compound heterozygous mutants c.1852_1854delAAG (p.K618del) and c.1852_1853delinsGC (p.K618A) in exon 16. In the MSH2 gene, we identified mutation c.638dupT (p.L213fs) in exon 3. Conclusions. This is the first report of mutations in MMR genes in Mexican patients with LS and these appear to be novel.

S1965 Novel Evidence for Soma-Wide MLH1 Hypermethylation and a Global Methylation Defect in Non-Familial Early-Onset Colorectal Cancer

Introduction: Recently soma-wide monoallelic germline epigenetic inactivation of MLH1 has been proposed as a potential mechanism for DNA mismatch repair (MMR) gene inactivation in some individuals with colorectal cancer (CRC). The present study aimed to investigate whether this methylation defect would be limited to the MLH1 promoter, or represents a broader epigenetic defect involving multiple genes. Methods: A 20 year old woman presented with a sigmoid colon cancer, and a negative family history for any cancer. Her tumor was evaluated for DNA mismatch repair (MMR) gene defects, and she was tested for germline mutations in MMR genes. Quantitative pyrosequencing and bisulfite sequencing of cloned PCR products was used to ascertain MLH1 methylation status in all three germ-layers (blood, hair and buccal tissues), as well as in her Epstein-Barr virus (EBV)-transformed lymphoblastoid cells. Genome wide/global methylation status for ~1500 genes/methylation loci was determined in the patients blood and controls (which included both parents, her sibling, and 3 unrelated controls) using Illumina GoldenGate methylation microarrays. Results: The patients tumor showed MSI and loss of expression of MLH1, PMS2 and MSH6 proteins. However, no germline mutations were detected in MLH1, MSH2 or MSH6. A single nucleotide polymorphism (SNP) permitted allele identification; 14%monoallelicMLH1 methylation was found in the tumor, but there was no allelic imbalance at MLH1 in the tumor. MLH1 methylation was observed in the patients blood (10%), EBV transformed lymphocytes (10%), buccal mucosa (22%) and hair follicles (48%). No evidence for MLH1 methylation was present in patients family members or any healthy control (all <1%). Interestingly, MLH1 methylation increased to 24% in the patients blood after 5-FU based chemotherapy treatment. Genome wide methylation analyses showed that aberrant methylation in the blood was not limited to MLH1, but included 43 additional hypermethylated genes (including RUNX3, IGF1, PPARγ and NOTCH4) and another 34 genes which were hypomethylated (including CDK2, STAT5 and IL10). Conclusions: This study reports the youngest patient with soma-wide MLH1 hypermethylation and CRC. The increase in MLH1 methylation following chemotherapy suggests that cells with methylated MLH1 promoters are relatively resistant to the toxic effects of this drug. More importantly, our observation that the epigenetic defects were not restricted to the MLH1 promoter suggests the possibility of a broader epigenetic defect in those individuals with soma-wide methylation of MLH1.