Resistance of mammalian cells to S(N)1-type methylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) generally arises through increased expression of methylguanine methyltransferase (MGMT), which reverts the cytotoxic O(6)-methylguanine ((Me)G) to guanine, or through inactivation of the mismatch repair (MMR) system, which triggers cell death through aberrant processing of (Me)G/T mispairs generated during DNA replication when MGMT capacity is exceeded. Given that MMR and (Me)G-detoxifying proteins are functionally conserved through evolution, and that MMR-deficient Escherichia coli dam(-) strains are also resistant to MNNG, the finding that MMR status did not affect the sensitivity of Saccharomyces cerevisiae to MNNG was unexpected. Because (Me)G residues in DNA trigger homologous recombination (HR), we wondered whether the efficient HR in S. cerevisiae might alleviate the cytotoxic effects of (Me)G processing. We now show that HR inactivation sensitizes S. cerevisiae to MNNG and that, as in human cells, defects in the MMR genes MLH1 and MSH2 rescue this sensitivity. Inactivation of the EXO1 gene, which encodes the only exonuclease implicated in MMR to date, failed to rescue the hypersensitivity, which implies that scExo1 is not involved in the processing of (Me)G residues by the S. cerevisiae MMR system.

The CpG island methylator phenotype (CIMP-high, CIMP1) is a distinct phenotype associated with microsatellite instability (MSI) and BRAF mutation in colon cancer. Recent evidence suggests the presence of KRAS mutation-associated CIMP subtype (CIMP-low, CIMP2). We used cluster analysis, principal component analysis (PCA), and structural equation modeling (SEM), a novel strategy, to decipher the correlation structure of CpG island hypermethylation. Using a database of 861 colon and rectal cancers, DNA methylation at 16 CpG islands [CACNA1G, CDKN2A (p16/ink4a), CHFR, CRABP1, HIC1, IGF2, IGFBP3, MGMT, MINT-1, MINT-31, MLH1, NEUROG1, p14 (CDKN2A/arf), RUNX3, SOCS1, and WRN] was quantified by real-time PCR. Tumors were categorized into three groups: Group 1 with wild-type KRAS/BRAF (N = 440); Group 2 with mutant KRAS and wild-type BRAF (N = 308); and Group 3 with wild-type KRAS and mutant BRAF (N = 107). Tumors with mutant KRAS/BRAF (N = 6) were excluded. In unsupervised hierarchical clustering analysis, all but six markers (CACNA1G, IGF2, RUNX3, MGMT, MINT-1, and SOCS1) were differentially clustered with CIMP-high and CIMP-low according to KRAS and BRAF status. In SEM, the correlation structures between CIMP, locus-specific CpG island methylation, and MSI differed according to KRAS and BRAF status, which was consistent with PCA results. In conclusion, KRAS and BRAF mutations appear to differentially influence correlation structure of CpG island methylation. Our novel data suggest two distinct perturbations, resulting in differential locus-specific propensity of CpG methylation.

During meiosis, homologous chromosomes (homologs) perform reciprocal exchanges (crossovers) at a high frequency. Crossovers display interference, i.e. their spacing is more even than would be expected if they were placed randomly along the chromosomes. Concomitantly with crossover formation, synaptonemal complexes (SCs) appear between homologs: each chromosome forms an axial structure, the axial element (AE); the AEs of homologs align, and numerous transverse filaments connect the AEs to form an SC. Both the AE and the SC have been implicated in the imposition of interference. We investigated whether intact AEs or SCs are required for crossover interference in the mouse, using a mutant lacking AE protein SYCP3, which displays structurally abnormal AEs and incomplete synapsis. We estimated the level of interference from the spacing of immunofluorescent MLH1 foci, which mark almost all crossover sites in the mouse, along the SCs. The levels of interference among MLH1 foci in wild-type and Sycp3(-/-) mice were comparable, implying that neither an intact AE structure nor full synapsis is required for wild-type levels of interference.

Acrolein (Acr), a ubiquitous environmental contaminant, is a human carcinogen. Acr can react with DNA to form mutagenic α- and γ-hydroxy-1, N(2)-cyclic propano-2'-deoxyguanosine adducts (α-OH-Acr-dG and γ-OH-Acr-dG). We demonstrate here that Acr-dG adducts can be efficiently repaired by the nucleotide excision repair (NER) pathway in normal human bronchial epithelia (NHBE) and lung fibroblasts (NHLF). However, the same adducts were poorly processed in cell lysates isolated from Acr-treated NHBE and NHLF, suggesting that Acr inhibits NER. In addition, we show that Acr treatment also inhibits base excision repair and mismatch repair. Although Acr does not change the expression of XPA, XPC, hOGG1, PMS2 or MLH1 genes, it causes a reduction of XPA, XPC, hOGG1, PMS2, and MLH1 proteins; this effect, however, can be neutralized by the proteasome inhibitor MG132. Acr treatment further enhances both bulky and oxidative DNA damage-induced mutagenesis. These results indicate that Acr not only damages DNA but can also modify DNA repair proteins and further causes degradation of these modified repair proteins. We propose that these two detrimental effects contribute to Acr mutagenicity and carcinogenicity.

OBJECTIVE

To investigate if analysis of genetic alterations in the main pathways involved in endometrioid type carcinogenesis (PI3K-AKT, Wnt/β-catenin, P53-activation and MSI) improves the current risk assessment based on clinicopathological factors.

METHODS

Formalin fixed paraffin embedded (FFPE) primary tumor samples of 65 patients with FIGO-stage I endometrioid type endometrial cancer (EEC) were selected from the randomized PORTEC-2 trial. Tumors were stained by immunohistochemistry for P53, PTEN and β-catenin. Tumor DNA was isolated for sequence analysis of TP53 (exons 4 to 8), hotspot mutation analysis of KRAS (exon 1) and PI3K (exon 9 and 20) and microsatellite-instability (MSI) analysis including MLH1 promotor-methylation status. Univariate and multivariate analyses for disease-free survival (DFS) using Cox regression models were performed.

RESULTS

P53 status (HR 6.7, 95%CI 1.75-26.0, p=0.006) and MSI were the strongest single genetic prognostic factors for decreased DFS, while high PI3K-AKT pathway activation showed a trend and β-catenin was not prognostic. The combination of multiple activated pathways was the most powerful prognostic factor for decreased DFS (HR 5.0; 95%CI 1.59-15.6 p=0.006). Multiple pathway activation, found in 8% of patients, was strongly associated with aggressive clinical course. In contrast, 40% of patients had no alterations in the investigated pathways and had a very low risk of disease progression.

CONCLUSIONS

Activation of multiple oncogenic pathways in EEC was the most powerful prognostic factor for decreased DFS, resulting in an individual risk assessment superior to the current approach based on clinicopathological factors.

OBJECTIVE

Constitutional MLH1 epimutations manifest as promoter methylation and silencing of the affected allele in normal tissues, predisposing to Lynch syndrome-associated cancers. This study investigated their frequency and inheritance.

METHODS

A total of 416 individuals with a colorectal cancer showing loss of MLH1 expression and without deleterious germline mutations in MLH1 were ascertained from the Colon Cancer Family Registry (C-CFR). Constitutive DNA samples were screened for MLH1 methylation in all 416 subjects and for promoter sequence changes in 357 individuals.

RESULTS

Constitutional MLH1 epimutations were identified in 16 subjects. Of these, seven (1.7%) had mono- or hemi-allelic methylation and eight had low-level methylation (2%). In one subject the epimutation was linked to the c.-27C>A promoter variant. Testing of 37 relatives from nine probands revealed paternal transmission of low-level methylation segregating with a c.+27G>A variant in one case. Five additional probands had a promoter variant without an MLH1 epimutation, with three showing diminished promoter activity in functional assays.

CONCLUSIONS

Although rare, sequence changes in the regulatory region of MLH1 and aberrant methylation may alone or together predispose to the development of cancer. Screening for these changes is warranted in individuals who have a negative germline sequence screen of MLH1 and loss of MLH1 expression in their tumor.Genet Med 2013:15(1):25-35.

This study examined how the DNA mismatch repair (MMR) system and p53 interact to maintain genomic integrity in the presence of the mutagenic stress induced by cisplatin (DDP). Sensitivity to the cytotoxic and mutagenic effect of DDP was assessed using a panel of sublines of the MMR-deficient HCT116 colon carcinoma cells in which MMR function had been restored by transfer of a copy of MLH1 on chromosome 3 or in which p53 function had been disabled by expression of HPV-16 E6. Loss of p53 function by expression of E6 in MMR-proficient HCT116+ ch3 cells conferred only 1.1-2.0-fold resistance to a panel of commonly used chemotherapeutic agents, whereas disruption of p53 in MMR-deficient HCT116 cells resulted in substantial levels of resistance to some agents (paclitaxel, 1.9-fold; gemcitabine, 2.7-fold; 6-thioguanine, 3.3-fold; and etoposide, 4.4-fold) but sensitization to other agents (topotecan, 2.5-fold; and DDP, 3.3-fold). Loss of MMR or p53 alone had only a minor effect on sensitivity to the mutagenic effect of DDP as measured by the appearance of variants resistant to 6-thioguanine, etoposide, topotecan, gemcitabine, and paclitaxel in the population 10 days later (1.0-2.4-fold), whereas loss of both p53 and MMR had a more profound effect (1.7-6.5-fold). Loss of both p53 and MMR increased the basal frequency insertion/deletion mutations detected by a shuttle vector-based assay to a greater extent than loss of either alone. In association with DDP-induced injury, loss of p53 or MMR alone resulted in 1.2- and 1.7-fold more mutations, whereas loss of both resulted in a 5.1-fold increase in mutant frequency. Examination of the impact of loss of p53 and/or MMR on the DDP-induced cell cycle checkpoint activation, p53 induction, ability of the cell to tolerate adducts in its DNA, and the rate of disappearance of platinum from genomic DNA indicated the effects of the loss of p53 and/or MMR on all of these parameters, suggesting a multifactorial etiology for the changes in sensitivity to the cytotoxic and mutagenic effects of DDP. These results indicate that p53 and MMR can cooperate to control sensitivity to the cytotoxic effect of DDP and to limit its mutagenic potential in the colon cancer cells.

BACKGROUND

The Muir-Torre syndrome (MTS) is an autosomal-dominant genodermatosis characterized by the presence of sebaceous gland tumors, with or without keratoacanthomas, associated with visceral malignancies. A subset of patients with MTS is considered a variant of the hereditary nonpolyposis colorectal carcinoma, which is caused by mutations in mismatch-repair genes. The objective of the current study was to evaluate whether a combined clinical, immunohistochemical, and biomolecular approach could be useful for the identification of Muir-Torre syndrome among patients with a diagnosis of sebaceous tumors and keratoacanthomas.

METHODS

The authors collected sebaceous skin lesions and keratoacanthomas recorded in the files of the Pathology Department of the University of Modena during the period 1986-2000. Through interviews and examination of clinical charts, family trees were drawn for 120 patients who were affected by these skin lesions.

RESULTS

Seven patients also were affected by gastrointestinal tumors, thus meeting the clinical criteria for the diagnosis of MTS. In the MTS families, a wide phenotypic variability was evident, both in the spectrum of visceral tumors and in the type of skin lesions. Microsatellite instability was found in five MTS patients: These patients showed concordance with immunohistochemical analysis; moreover, a constitutional mutation in the MSH2 gene was found in 1 patient. Lack of expression of MSH2/MSH6 or MLH1 proteins was evident in the skin lesions and in the associated internal malignancies of 3 patients and 2 patients with MTS, respectively.

CONCLUSIONS

The clinical, biomolecular, and immunohistochemical characterization of sebaceous skin lesions and keratoacanthomas may be used as screening for the identification of families at risk of MTS, a disease that is difficult to recognize and diagnose.

The hPMS2 mutation E705K is associated with Turcot syndrome. To elucidate the pathogenesis of hPMS2-E705K, we modeled this mutation in yeast and characterized its expression and effects on mutation avoidance in mammalian cells. We found that while hPMS2-E705K (pms1-E738K in yeast) did not significantly affect hPMS2 (Pms1p in yeast) stability or interaction with MLH1, it could not complement the mutator phenotype in MMR-deficient mouse or yeast cells. Furthermore, hPMS2-E705K/pms1-E738K inhibited MMR in wild-type (WT) mammalian cell extracts or yeast cells only when present in excess amounts relative to WT PMS2. Our results strongly suggest that hPMS2-E705K is a recessive loss-of-function allele.

The mismatch repair (MMR) family complexes Msh4-Msh5 and Mlh1-Mlh3 act with Exo1 and Sgs1-Top3-Rmi1 in a meiotic double strand break repair pathway that results in the asymmetric cleavage of double Holliday junctions (dHJ) to form crossovers. This review discusses how meiotic roles for Msh4-Msh5 and Mlh1-Mlh3 do not fit paradigms established for post-replicative MMR. We also outline models used to explain how these factors promote the formation of meiotic crossovers required for the accurate segregation of chromosome homologs during the Meiosis I division.

Background: Although high-risk mutations in identified major susceptibility genes (DNA mismatch repair genes and MUTYH) account for some familial aggregation of colorectal cancer, their population prevalence and the causes of the remaining familial aggregation are not known.Methods: We studied the families of 5,744 colorectal cancer cases (probands) recruited from population cancer registries in the United States, Canada, and Australia and screened probands for mutations in mismatch repair genes and MUTYH We conducted modified segregation analyses using the cancer history of first-degree relatives, conditional on the proband's age at diagnosis. We estimated the prevalence of mutations in the identified genes, the prevalence of HR for unidentified major gene mutations, and the variance of the residual polygenic component.Results: We estimated that 1 in 279 of the population carry mutations in mismatch repair genes (MLH1 = 1 in 1,946, MSH2 = 1 in 2,841, MSH6 = 1 in 758, PMS2 = 1 in 714), 1 in 45 carry mutations in MUTYH, and 1 in 504 carry mutations associated with an average 31-fold increased risk of colorectal cancer in unidentified major genes. The estimated polygenic variance was reduced by 30% to 50% after allowing for unidentified major genes and decreased from 3.3 for age <40 years to 0.5 for age ≥70 years (equivalent to sibling relative risks of 5.1 to 1.3, respectively).Conclusions: Unidentified major genes might explain one third to one half of the missing heritability of colorectal cancer.Impact: Our findings could aid gene discovery and development of better colorectal cancer risk prediction models. Cancer Epidemiol Biomarkers Prev; 26(3); 404-12. ©2016 AACR.

Serrated polyps (hyperplastic polyps, sessile or traditional serrated adenomas), which can arise in a sporadic or polyposis setting, predispose to colorectal cancer (CRC), especially those with microsatellite instability (MSI) due to MLH1 promoter methylation (MLH1me+). We investigate genetic alterations in the serrated polyposis pathway.

We used a combination of exome sequencing and target gene Sanger sequencing to study serrated polyposis families, sporadic serrated polyps and CRCs, with validation by analysis of The Cancer Genome Atlas (TCGA) cohort, followed by organoid-based functional studies.

In one out of four serrated polyposis families, we identified a germline RNF43 mutation that displayed autosomal dominant cosegregation with the serrated polyposis phenotype, along with second-hit inactivation through loss of heterozygosity or somatic mutations in all serrated polyps (16), adenomas (5) and cancer (1) examined, as well as coincidental BRAF mutation in 62.5% of the serrated polyps. Concurrently, somatic RNF43 mutations were identified in 34% of sporadic sessile/traditional serrated adenomas, but 0% of hyperplastic polyps (p=0.013). Lastly, in MSI CRCs, we found significantly more frequent RNF43 mutations in the MLH1me+ (85%) versus MLH1me- (33.3%) group (p<0.001). These findings were validated in the TCGA MSI CRCs (p=0.005), which further delineated a significant differential involvement of three Wnt pathway genes between these two groups (RNF43 in MLH1me+; APC and CTNNB1 in MLH1me-); and identified significant co-occurrence of BRAF and RNF43 mutations in the MSI (p<0.001), microsatellite stable (MSS) (p=0.002) and MLH1me+ MSI CRCs (p=0.042). Functionally, organoid culture of serrated adenoma or mouse colon with CRISPR-induced RNF43 mutations had reduced dependency on R-spondin1.

These results illustrate the importance of RNF43, along with BRAF mutation in the serrated neoplasia pathway (both the sporadic and familial forms), inform genetic diagnosis protocol and raise therapeutic opportunities through Wnt inhibition in different stages of evolution of serrated polyps.

BACKGROUND

Although the essential roles of stromata in tumor development have been recognized, the morphologic classification of desmoplastic reaction (DR) in colorectal cancer (CRC) is unclear.

METHODS

In this study, DRs were histologically classified into three patterns based on the products of activated fibroblasts (i.e., keloid-like collagen and myxoid stroma). The prognostic impact of DRs was evaluated in two independent cohorts of stages 2 and 3 CRC patients: cohort 1 (880 patients) and cohort 2 (474 patients). The association of DR and the local environment was investigated immunohistochemically.

RESULTS

In cohort 1, mature DR was shown by 413 patients, intermediate DR by 275 patients, and immature DR by 192 patients. Categorization of DR was significantly associated with tumor location, pT and pN stages, tumor differentiation, venous invasion, tumor budding, and Crohn's-like lymphoid reaction (P ≤ 0.0001-0.008). Immature DR was relevant to the high incidence of recurrence in the liver, lung, lymph nodes, peritoneum, and locoregional areas (P ≤ 0.0001-0.002). The 5-year disease-free survival rate was highest in the mature group (87 %), followed by the intermediate group (72 %) and the immature group (49 % (P < 0.0001). In the multivariate analysis, DR showed an impact on survival outcome independent of conventional prognostic factors, including pT and pN stages. These results were similarly observed in cohort 2. Immature DR was associated with normal MutL homologue 1 (MLH1)/MutS homologue 2 (MSH2) immunoreactivity, a smaller number of infiltrating CD8(+) T cells and tumor-associated macrophages, a decreased microvessel count, and positive expression of tenascin-C and fibronectin.

CONCLUSIONS

The proposed histologic DR categorization directly reflects tumor behavior in a modulating stromal environment and could provide valuable prognostic information for CRC patients.

Epigenetic silencing of cancer-related genes by promoter methylation is a frequent event in sporadic colorectal cancer. The CpG island methylator phenotype (CIMP+), in which discrete genes throughout the genome are simultaneously methylated, and long-range epigenetic silencing, whereby multiple genes within contiguous chromosomal regions are methylated, have been described in subsets of colorectal cancer. We previously reported the concurrent methylation of the mismatch repair gene MLH1 with a cluster of flanking genes in chromosome region 3p22 in sporadic colorectal carcinoma exhibiting microsatellite instability and the BRAF V600E mutation. Herein, we aimed to determine whether methylation of MLH1 and neighbouring 3p22 genes, singly or concomitantly, correlate with the germline c.-93G>A SNP within the MLH1 promoter, CIMP+ and other clinicopathological and molecular features of the tumours. By studying a cohort of 946 sporadic colorectal cancer cases, we show a strong association between concordant methylation of ≥ 3 of five 3p22 genes with CIMP+ and the BRAF V600E mutation (P<0.001). These associations were independent of microsatellite instability, as concomitant methylation of 3p22 genes other than MLH1 was found in microsatellite stable cancers. These findings show that long-range epigenetic silencing across 3p22 occurs in the context of CIMP+ and the BRAF V600E mutation, and only gives rise to microsatellite instability when this process encompasses MLH1. Furthermore, the strong relationship between long-range epigenetic silencing of 3p22 and CIMP+ provides further evidence that these two purportedly distinct epigenetic phenotypes represent a single entity with a common aetiology. Low-level methylation of MLH1 and flanking 3p22 genes, as well as the BRAF V600E mutation, were detected in the apparently normal colonic mucosa of a small number of cases whose tumours showed a similar molecular profile, suggesting that these concurring genetic and epigenetic events can occur as a field defect in neoplastic development.

The microsatellite instability (MSI) pathway is one of the important mutational pathways that play a critical role in colorectal carcinogenesis. About 15% of colorectal cancers (CRCs) are characterized by MSI. MSI tumors usually arise because of a genetic defect in mismatch repair (MMR) genes, one of the main DNA-repairing systems. MMR is a highly conserved biological pathway that plays a key role in maintaining genomic stability by correcting the base-base mismatches and insertion/deletion mispairs generated during DNA replication and recombination. Escherichia coli MutS and MutL and their eukaryotic homologs, MutSα and MutLα, respectively, are key players in MMR-associated genome maintenance. Mutations in at least five pivotal genes of MMR, namely, in those encoding mutS homolog 2 (MSH2), mutL homolog 1 (MLH1), mutS homolog 6 (MSH6), postmeiotic segregation increased 1 (PMS1), and postmeiotic segregation, increased 2 (PMS2) have been found in CRC, highlighting the importance of understanding the basic structure and functions of the essential molecules that make up the MMR system. In this review, we have attempted to focus on this aspect, that is, the role that MMR molecules play in CRC carcinogenesis.

Sessile serrated adenomas (SSAs) are the precursors of at least 15% of colorectal carcinomas, but their biology is incompletely understood. We performed a clinicopathological and molecular analysis of a large number of the rarely observed SSAs with dysplasia/carcinoma to better define their features and the pathways by which they progress to carcinoma.

A cross-sectional analysis of 137 SSAs containing regions of dysplasia/carcinoma prospectively collected at a community GI pathology practice was conducted. Samples were examined for BRAF and KRAS mutations, the CpG island methylator phenotype (CIMP) and immunostained for MLH1, p53, p16, β-catenin and 0-6-methylguanine DNA methyltransferase (MGMT).

The median polyp size was 9 mm and 86.5% were proximal. Most were BRAF mutated (92.7%) and 94.0% showed CIMP. Mismatch repair deficiency, evidenced by loss of MLH1 (74.5%) is associated with older age (76.7 versus 71.0; p<0.0029), female gender (70% versus 36%; p<0.0008), proximal location (91% versus 72%; p<0.02), CIMP (98% versus 80%; p<0.02) and lack of aberrant p53 (7% versus 34%; p<0.001) when compared with the mismatch repair-proficient cases. Loss of p16 (43.1%) and gain of nuclear β-catenin (55.5%) were common in areas of dysplasia/cancer, irrespective of mismatch repair status.

SSAs containing dysplasia/carcinoma are predominantly small (<10 mm) and proximal. The mismatch repair status separates these lesions into distinct clinicopathological subgroups, although WNT activation and p16 silencing are common to both. Cases with dysplasia occur at a similar age to cases with carcinoma. This, together with the rarity of these 'caught in the act' lesions, suggests a rapid transition to malignancy following a long dwell time as an SSA without dysplasia.

Undifferentiated gastrointestinal tract carcinomas are rare highly aggressive neoplasms with frequent but not obligatory rhabdoid features. Recent studies showed loss of SMARCB1 (INI1), a core subunit of the SWI/SNF chromatin remodeling complex, in 50% of tested cases. However, the molecular pathways underlying histologically similar but SMARCB1-intact cases are unknown. We herein analyzed 13 cases for expression of 4 SWI/SNF complex subunits SMARCB1, SMARCA2, SMARCA4, and ARID1A and the mismatch-repair proteins MLH1, MSH2, MSH6, and PMS2 by immunohistochemistry. Patients included 12 men and 1 woman aged 32 to 81 years (median, 57 y). Site of origin was colon (5), small bowel (2), stomach (3), small+large intestine (1), small intestine+ampulla of Vater (1), and esophagogastric junction (1). All tumors showed anaplastic large to medium-sized cells with variable rhabdoid features, pleomorphic giant cells, and, rarely, spindle cell foci. Abortive gland formation was seen in 3 cases and bona fide glandular component in 1 case. Most cases strongly expressed vimentin and variably pancytokeratin. In total, 12/13 cases (92%) showed loss of at least 1 SWI/SNF component. Loss of SMARCB1 (5/13), SMARCA2 (10/13), SMARCA4 (2/13), and ARID1A (2/13) was observed either in combination or isolated. SMARCA2 loss was isolated in 5 cases and coexisted with lost SMARCB1 in 5 cases (all 5 SMARCB1-deficient tumors showed loss of SMARCA2 as well). Co-inactivation of SMARCB1 and SMARCA4 or of SMARCA2 and SMARCA4 was not observed. Two mismatch-repair-deficient cases (MLH1/PMS2) showed concurrent loss of SMARCB1, SMARCA2, and (one of them) ARID1A. This study illustrates for the first time loss of different components of the SWI/SNF complex other than SMARCB1 in undifferentiated gastrointestinal carcinomas including novel SMARCA4-deficient and SMARCA2-deficient cases. Our results underline the close link between SWI/SNF deficiency and the aggressive rhabdoid phenotype. Frequent loss of SMARCA2 possibly points to fragility/vulnerability of the SWI/SNF complex as a consequence of lost core subunit SMARCB1. The exact molecular mechanisms underlying co-inactivation of different SWI/SNF subunits merit further investigations.

Hereditary non-polyposis colorectal cancer (HNPCC) is an autosomal-dominant disorder characterized by predisposition to colorectal cancer and extracolonic malignancies, frequent multiple primary tumors in the same patient, and early age of cancer onset. A main clinical variant of Lynch syndrome, Muir-Torre syndrome (MTS) is characterized by the association between one or more visceral malignancies, with at least one sebaceous skin tumor or keratoacanthoma. In our study, we have screened a cohort of 538 HNPCC patients, related to 57 HNPCC families, to detect sebaceous skin tumors and keratoacanthomas and the role of mismatch repair (MMR) genes, MLH1, MSH2, and MSH6, in their pathogenesis. Among the 57 HNPCC families, we have identified four MTS families and one suspected MTS family, in which sebaceous carcinoma was found in one HNPCC mutation carrier subject who did not show visceral malignancy. In four of these families, linked to two MLH1 mutations and to two MSH2 mutations, biomolecular characterization showed concordance among immunohistochemistry analysis and gene mutations. The evidences of our investigations show that MLH1 and MSH2 gene mutations have an equivalent etiopathological role both for Lynch syndrome and for MTS; hence, we propose a broadened clinical criteria for definition of Lynch syndrome that will include sebaceous adenoma, carcinoma, and keratoacanthoma.

Genetic background effects underlie the penetrance of most genetically determined phenotypes, including human diseases. To explore how such effects can modify a mutant phenotype in a genetically tractable system, we examined an incompatibility involving the MLH1 and PMS1 mismatch repair genes using a large population sample of geographically and ecologically diverse Saccharomyces cerevisiae strains. The mismatch repair incompatibility segregates into naturally occurring yeast strains, with no strain bearing the deleterious combination. In assays measuring the mutator phenotype conferred by different combinations of MLH1 and PMS1 from these strains, we observed a mutator phenotype only in combinations predicted to be incompatible. Surprisingly, intragenic modifiers could be mapped that specifically altered the strength of the incompatibility over a 20-fold range. Together, these observations provide a powerful model in which to understand the basis of disease penetrance and how such genetic variation, created through mating, could result in new mutations that could be the raw material of adaptive evolution in yeast populations.

In mismatch repair (MMR), members of the MLH gene family have been proposed to act as key molecular matchmakers to coordinate mismatch recognition with downstream repair functions that result in mispair excision. Two members of this gene family, MLH1 and MLH3, have also been implicated in meiotic crossing over. These diverse roles suggest that a mutational analysis of MLH genes could provide reagents required to identify interactions between gene products and to test whether the different roles ascribed to a subset of these genes can be separated. In this report we show that in Saccharomyces cerevisiae the mlh1Delta mutation confers inviability in pol3-01 strain backgrounds that are defective in the Poldelta proofreading exonuclease activity. This phenotype was exploited to identify four mlh1 alleles that each confer a temperature-sensitive phenotype for viability in pol3-01 strains. In three different mutator assays, strains bearing conditional mlh1 alleles displayed wild-type or nearly wild-type mutation rates at 26 degrees. At 35 degrees, these strains exhibited mutation rates that approached those observed in mlh1Delta mutants. The mutator phenotype exhibited in mlh1-I296S strains was partially suppressed at 35 degrees by EXO1 overexpression. The mlh1-F228S and -I296S mutations conferred a separation-of-function phenotype in meiosis; both mlh1-F228S and -I296S strains displayed strong defects in meiotic mismatch repair but showed nearly wild-type levels of crossing over, suggesting that the conditional mutations differentially affected MLH1 functions. These genetic studies suggest that the conditional mlh1 mutations can be used to separate the MMR and meiotic crossing-over functions of MLH1 and to identify interactions between MLH1 and downstream repair components.

BACKGROUND

Molecular testing for hereditary nonpolyposis colorectal carcinoma (HNPCC) is becoming standard care and it is cost-effective compared with no genetic testing. However, the best strategy for detection of HNPCC gene carriers is unknown.

METHODS

We use a decision analytic model to evaluate the effectiveness and incremental cost-effectiveness of four commonly used testing strategies to detect HNPCC gene carriers. The model starts with a population of colorectal carcinoma (CRC) patients and measures costs, the number of gene carriers detected, and incremental costs per gene carrier detected.

RESULTS

We found that germline testing on only those CRC probands who meet the Amsterdam criteria detects the fewest gene carriers and has the lowest cost whereas tumor microsatellite instability (MSI) testing of all CRC patients and families has the highest cost and detects the most gene carriers. When cost-effectiveness is considered, the mixed strategy (MSH2 and MLH1 testing on those who meet the Amsterdam criteria and germline testing for the remainder who meet less stringent modified criteria and are MSI-High) seems superior. The mixed strategy detects 59.6 mutation carriers per 1000 CRC cases and costs much less than the test all strategy, which has an incremental cost-effectiveness of $51,151. The mixed strategy often other strategies and when compared to the Amsterdam strategy, has a cost-effectiveness of only $6441 per gene carrier detected.

CONCLUSIONS

It is not very effective to limit genetic testing to only individuals who meet the Amsterdam criteria, as many gene carriers are missed. However, testing all CRC patients for tumor MSI-H, although effective, may be prohibitively expensive. A mixed strategy is the more cost-effective approach.

BACKGROUND

Given the high incidence of metastatic esophageal squamous cell carcinoma, especially in Asia, we screened for the presence of somatic mutations using OncoMap platform with the aim of defining subsets of patients who may be potential candidate for targeted therapy.

METHODS

We analyzed 87 tissue specimens obtained from 80 patients who were pathologically confirmed with esophageal squamous cell carcinoma and received 5-fluoropyrimidine/platinum-based chemotherapy. OncoMap 4.0, a mass-spectrometry based assay, was used to interrogate 471 oncogenic mutations in 41 commonly mutated genes. Tumor specimens were prepared from primary cancer sites in 70 patients and from metastatic sites in 17 patients. In order to test the concordance between primary and metastatic sites from the patient for mutations, we analyzed 7 paired (primary-metastatic) specimens. All specimens were formalin-fixed paraffin embedded tissues and tumor content was >70%.

RESULTS

In total, we have detected 20 hotspot mutations out of 80 patients screened. The most frequent mutation was PIK3CA mutation (four E545K, five H1047R and one H1047L) (N = 10, 11.5%) followed by MLH1 V384D (N = 7, 8.0%), TP53 (R306, R175H and R273C) (N = 3, 3.5%), BRAF V600E (N = 1, 1.2%), CTNNB1 D32N (N = 1, 1.2%), and EGFR P733L (N = 1, 1.2%). Distributions of somatic mutations were not different according to anatomic sites of esophageal cancer (cervical/upper, mid, lower). In addition, there was no difference in frequency of mutations between primary-metastasis paired samples.

CONCLUSIONS

Our study led to the detection of potentially druggable mutations in esophageal SCC which may guide novel therapies in small subsets of esophageal cancer patients.

BACKGROUND

We modified a tetracycline-regulated system that can control the activity of individual genes quantitatively and reversibly in transgenic mammals. Despite these advances, there remained one problem in the intensive use of the tet-system: the limited range of acceptor cell lines, expressing a tetracycline-controlled transcriptional activator (tTA). This study describes in detail new vectors and a unifying strategy to generate tTA-expressing cell lines.

METHODS

Two retroviral vectors pLNCtTA-hCMV and pLNCtTA-EF1alpha coding for the tTA were used to engineer cell lines to constitutively express tTA. New expression vectors pETE-Hyg and pETE-Bsd were also created that replicate in episomal form in human cells and facilitate tetracycline-regulated expression of targeted genes.

RESULTS

The primate-tropic retroviruses efficiently delivered the regulatory tTA gene into 12 selected human cancer cell lines. Two candidate tumor suppressor genes from the human 3p21-p22 region MAPKAPK3 (3pK) and MLH1 were cloned into the episomal vector and transfected into engineered A9 and KRC/Y cells. The transfectants were subcutaneously grown in SCID mice, and the expression of the transgene was successfully controlled in vivo by tetracycline administered ad libitum in drinking water. The experiments demonstrated that both transgenes did not antagonize the tumorous growth of these cells.

CONCLUSIONS

New retroviral and episomal vectors appear particularly suited for tight regulation of genes that cause suppression of cell growth. The generated cell lines can be used in various applications to study the effect of an inducible transgene in human cancer cells.

Defects in DNA mismatch repair (MMR) are common in human cancers, confer tolerance to certain types of chemotherapeutic agents, and lead to genomic instability. In addition to their mismatch-correcting roles during DNA replication, MMR proteins can bind to certain DNA lesions and signal p53 and apoptosis by an unknown mechanism. To further study the mechanism by which the MMR protein MLH1 is involved in the induction of p53 and apoptosis, we exposed the colon carcinoma cell line HCT116 (MLH1-deficient) and mlh1-corrected HCT116 sublines to alkylating agents or hydrogen peroxide (H2O2). It was found that while alkylating agents induced both apoptosis and phosphorylation of the Ser-15 site of p53 in a MLH1-dependent manner, induction of apoptosis, but not p53 phosphorylation, was MLH1 dependent following treatment with H2O2. The MLH1-dependent induction of p53 phosphorylation by alkylating agents did not appear to be cell cycle dependent, arguing against a futile repair mechanism operating during S phase as the sole mechanism for the MLH1-dependent DNA damage signaling. Importantly, we found that both alkylating agents and H2O2 caused significant inhibition of mRNA synthesis in MLH1-expressing but not in MLH1-deficient cells. These findings suggest a novel mechanism of MLH1 in the induction p53 and apoptosis by inhibiting RNA polymerase II-dependent transcription on damaged DNA templates.