MSH4 is a meiosis-specific MutS homolog. In yeast, it is required for reciprocal recombination and proper segregation of homologous chromosomes at meiosis I. MLH1 (MutL homolog 1) facilitates both mismatch repair and crossing over during meiosis in yeast. Germ-line mutations in the MLH1 human gene are responsible for hereditary nonpolyposis cancer, but the analysis of MLH1-deficient mice has revealed that MLH1 is also required for reciprocal recombination in mammals. Here we show that hMSH4 interacts with hMLH1. The two proteins are coimmunoprecipitated regardless of the presence of DNA or ATP, suggesting that the interaction does not require the binding of MSH4 to DNA. The domain of hMSH4 responsible for the interaction is in the amino-terminal part of the protein whereas the region that contains the ATP binding site and helix-turn-helix motif does not bind to hMLH1. Immunolocalization analysis shows that MSH4 is present at sites along the synaptonemal complex as soon as homologous chromosomes synapse. The number of MSH4 foci decreases gradually as pachynema progresses. During this transition, MLH1 foci begin to appear and colocalize with MSH4. These results suggest that MSH4 is first required for chromosome synapsis and that this MutS homologue is involved later with MLH1 in meiotic reciprocal recombination.

OBJECTIVE

The aim of this study is to evaluate if mismatch repair (MMR) defective colorectal cancer has a different response to adjuvant 5-fluorouracil (5-FU) chemotherapy in a cohort of patients prospectively followed during 5 years.

METHODS

The cohort included 754 surgically treated patients with colorectal cancer. MMR status was diagnosed by MLH1 and MSH2 immunohistochemistry and microsatellite instability analysis. Median follow-up was 49.2 months (range 1-73). At inclusion, 505 patients were diagnosed as TNM II or III stage, analysis of the efficacy of adjuvant chemotherapy was made on this population. Adjuvant chemotherapy was applied to 248 patients (98.2% 5-FU based).

RESULTS

MMR deficiency was found in 76 patients (10.1%). No differences were found in overall survival (log-rank p=0.3) or disease-free survival (log-rank p=0.3) regarding MMR status. Adjuvant chemotherapy improves survival in patients in the II or III stage, but this improvement is only evident in patients with MMR-competent tumours (log-rank p=0.00001). Survival of patients with MMR-defective tumours does not improve with adjuvant chemotherapy (log-rank p=0.7). A multivariate analysis showed an independent effect of the interaction between MMR status and adjuvant chemotherapy (Hazard ratio 2.04; 95% confidence interval: 1.42-2.93).

CONCLUSIONS

In a cohort of colorectal cancer patients, those with MMR-deficient tumours seem not to benefit from 5-FU-based chemotherapy.

BACKGROUND

Endometrial carcinoma is a common malignancy in hereditary nonpolyposis colorectal carcinoma (HNPCC). Like colon carcinoma, endometrial carcinoma is diagnosed at an earlier age in women with HNPCC. In contrast to colon carcinoma, the pathologic features of endometrial carcinoma in HNPCC have not been studied in detail. It was the purpose of this study to pathologically characterize a series of HNPCC associated endometrial carcinomas.

METHODS

Fifty women with HNPCC and endometrial carcinoma were analyzed from four different hereditary cancer registries. H&E stained slides and pathology reports were reviewed for clinically important pathologic features of endometrial carcinoma. These results were compared with those for two different groups of sporadic endometrial carcinoma--women younger than age 50 years (n = 42) and women of all ages with tumors demonstrating microsatellite instability (MSI-high) secondary to methylation of MLH1 (n = 26).

RESULTS

Nearly one-fourth of HNPCC patients in this study had endometrial tumors with pathologic features that would require adjuvant therapy after hysterectomy. There was a trend toward the HNPCC patients having more nonendometrioid tumors; all of these patients were carriers of MSH2 mutations. Such nonendometrioid tumors were extremely rare in the MLH1 methylated group. A subset of MLH1 methylated sporadic tumors demonstrated a unique, 'undifferentiated' histology that was not observed in HNPCC or the young group.

CONCLUSIONS

Data suggest a genotype-phenotype relation in which microsatellite instability resulting from MLH1 methylation is almost exclusively associated with classical or 'undifferentiated' endometrioid tumors, whereas microsatellite instability secondary to MSH2 mutation can result in a more variable histologic spectrum of endometrial carcinoma.

Mbd4 (methyl-CpG binding domain 4) is a novel mammalian repair enzyme that has been implicated biochemically in the repair of mismatched G-T residues at methylated CpG sites. In addition, the human protein has been shown to interact with the DNA mismatch repair protein MLH1. To clarify the role of Mbd4 in DNA repair in vivo and to examine the impact of Mbd4 inactivation on gastrointestinal (GI) tumorigenesis, we introduced a null mutation into the murine Mbd4 gene by gene targeting. Heterozygous and homozygous Mbd4 mutant mice develop normally and do not show increased cancer susceptibility or reduced survival. Although Mbd4 inactivation did not increase microsatellite instability (MSI) in the mouse genome, it did result in a 2- to 3-fold increase in C->>T transition mutations at CpG sequences in splenocytes and epithelial cells of the small intestinal mucosa. The combination of Mbd4 deficiency with a germ line mutation in the adenomatous polyposis coli (Apc) gene increased the tumor number in the GI tract and accelerated tumor progression. The change in the GI cancer phenotype was associated with an increase in somatic C->>T mutations at CpG sites within the coding region of the wild-type Apc allele. These studies indicate that, although inactivation of Mbd4 does not by itself cause cancer predisposition in mice, it can alter the mutation spectrum in cancer cells and modify the cancer predisposition phenotype.

Eukaryotic genomes contain tracts of DNA in which a single base or a small number of bases are repeated (microsatellites). Mutations in the yeast DNA mismatch repair genes MSH2, PMS1, and MLH1 increase the frequency of mutations for normal DNA sequences and destabilize microsatellites. Mutations of human homologs of MSH2, PMS1, and MLH1 also cause microsatellite instability and result in certain types of cancer. We find that a mutation in the yeast gene MSH3 that does not substantially affect the rate of spontaneous mutations at several loci increases microsatellite instability about 40-fold, preferentially causing deletions. We suggest that MSH3 has different substrate specificities than the other mismatch repair proteins and that the human MSH3 homolog (MRP1) may be mutated in some tumors with microsatellite instability.

Previous studies from our laboratory indicated that expression of the MLH1 DNA mismatch repair (MMR) gene was necessary to restore cytotoxicity and an efficient G(2) arrest in HCT116 human colon cancer cells, as well as Mlh1(-/-) murine embryonic fibroblasts, after treatment with 5-fluoro-2'-deoxyuridine (FdUrd). Here, we show that an identical phenomenon occurred when expression of MSH2, the other major MMR gene, was restored in HEC59 human endometrial carcinoma cells or was present in adenovirus E1A-immortalized Msh2(+/+) (compared with isogenic Msh2(-/-)) murine embryonic stem cells. Because MMR status had little effect on cellular responses (i.e. G(2) arrest and lethality) to the thymidylate synthase inhibitor, Tomudex, and a greater level of [(3)H]FdUrd incorporation into DNA was found in MMR-deficient cells, we concluded that the differential FdUrd cytotoxicity between MMR-competent and MMR-deficient cells was mediated at the level of DNA incorporation. Analyses of ATPase activation suggested that the hMSH2-hMSH6 heterodimer only recognized FdUrd moieties (as the base 5-fluorouracil (FU) in DNA) when mispaired with guanine, but not paired with adenine. Furthermore, analyses of incorporated FdUrd using methyl-CpG-binding domain 4 glycosylase indicated that there was more misincorporated FU:Gua in the DNA of MMR-deficient HCT116 cells. Our data provide the first demonstration that MMR specifically detects FU:Gua (in the first round of DNA replication), signaling a sustained G(2) arrest and lethality.

DNA mismatch repair is thought to act through two subpathways involving the recognition of base-base and insertion/deletion mispairs by the Msh2-Msh6 heterodimer and the recognition of insertion/deletion mispairs by the Msh2-Msh3 heterodimer. Here, through genetic and biochemical approaches, we describe a previously unidentified role of the Msh2-Msh3 heterodimer in the recognition of base-base mispairs and the suppression of homology-mediated duplication and deletion mutations. Saccharomyces cerevisiae msh3 mutants did not show an increase in the rate of base substitution mutations by the CAN1 forward mutation assay compared to the rate for the wild type but did show an altered spectrum of base substitution mutations, including an increased accumulation of base pair changes from GC to CG and from AT to TA; msh3 mutants also accumulated homology-mediated duplication and deletion mutations. The mutation spectrum of mlh3 mutants paralleled that of msh3 mutants, suggesting that the Mlh1-Mlh3 heterodimer may also play a role in the repair of base-base mispairs and in the suppression of homology-mediated duplication and deletion mutations. Mispair binding analysis with purified Msh2-Msh3 and DNA substrates derived from CAN1 sequences found to be mutated in vivo demonstrated that Msh2-Msh3 exhibited robust binding to specific base-base mispairs that was consistent with functional mispair binding.

OBJECTIVE

We investigated the prevalence of germline mutations in APC, ATM, BRCA1, BRCA2, CDKN2A, MLH1, MSH2, MSH6, PALB2, PMS2, PRSS1, STK11, and TP53 in patients with pancreatic cancer.

METHODS

The Ontario Pancreas Cancer Study enrolls consenting participants with pancreatic cancer from a province-wide electronic pathology database; 708 probands were enrolled from April 2003 through August 2012. To improve the precision of BRCA2 prevalence estimates, 290 probands were selected from 3 strata, based on family history of breast and/or ovarian cancer, pancreatic cancer, or neither. Germline DNA was analyzed by next-generation sequencing using a custom multiple-gene panel. Mutation prevalence estimates were calculated from the sample for the entire cohort.

RESULTS

Eleven pathogenic mutations were identified: 3 in ATM, 1 in BRCA1, 2 in BRCA2, 1 in MLH1, 2 in MSH2, 1 in MSH6, and 1 in TP53. The prevalence of mutations in all 13 genes was 3.8% (95% confidence interval, 2.1%-5.6%). Carrier status was associated significantly with breast cancer in the proband or first-degree relative (P < .01), and with colorectal cancer in the proband or first-degree relative (P < .01), but not family history of pancreatic cancer, age at diagnosis, or stage at diagnosis. Of patients with a personal or family history of breast and colorectal cancer, 10.7% (95% confidence interval, 4.4%-17.0%) and 11.1% (95% confidence interval, 3.0%-19.1%) carried pathogenic mutations, respectively.

CONCLUSIONS

A small but clinically important proportion of pancreatic cancer is associated with mutations in known predisposition genes. The heterogeneity of mutations identified in this study shows the value of using a multiple-gene panel in pancreatic cancer.

Heterozygous germ-line mutations in the DNA mismatch repair genes lead to hereditary nonpolyposis colorectal cancer. The disease susceptibility of individuals who constitutionally lack both wild-type alleles is unknown. We have identified three offspring in a hereditary nonpolyposis colorectal cancer family who developed hematological malignancy at a very early age, and at least two of them displayed signs of neurofibromatosis type 1 (NF1). DNA sequence analysis and allele-specific amplification in two siblings revealed a homozygous MLH1 mutation (C676T->>Arg226Stop). Thus, a homozygous germ-line MLH1 mutation and consequent mismatch repair deficiency results in a mutator phenotype characterized by leukemia and/or lymphoma associated with neurofibromatosis type 1.

The concept that cells can become malignant upon the elimination of parts of chromosomes inhibiting cell division dates back to Boveri in 1914. Deletions occurring in tumor cells are therefore considered a first indication of possible locations of tumor suppressor gene. Approaches used to localize and identify the paradigm of tumor suppressors, RB1, have also been applied to localize tumor suppressor genes on 3p, the short arm of chromosome 3. This review discusses the methodological advantages and limitations of the various approaches. From a review of the literature on losses of 3p in different types of solid tumors it appears that some tumor types show involvement of the same region, while between others the regions involved clearly differ. Also discussed are results of functional assays of tumor suppression by transfer of part of chromosome 3 into tumor cell lines. The likelihood that a common region of deletions would contain a tumor suppressor is strongly enhanced by coincidence of that region with a chromosome fragment suppressing tumorigenicity upon introduction in tumor cells. Such a situation exists for a region in 3p21.3 as well as for one or more in 3p12-p14. The former region is considered the location of a lung cancer suppressor. The same gene or a different one in the same region may also play a role in the development of other cancers including renal cell cancer. In the latter cancer, there may be additional roles of the VHL region and/or a 3p12-p14 region. The breakpoint region of a t(3;8) originally found to be constitutively present in a family with hereditary renal cell cancer now seems to be excluded from such a role. Specific genes on 3p have been suggested to act as suppressor genes based on either their location in a common deletion region, a markedly reduced expression or presence of aberrant transcripts, their capacity to suppress tumorigenicity upon transfection in to tumor cells, the presumed function of the gene product, or a combination of several of these criteria. A number of genes are evaluated for their possible role as a tumor suppressor according to these criteria. General agreement on such a role seems to exist only for VHL. Though hMLH1 plays an obvious role in the development of specific mismatch repair-deficient cancers, it cannot revert the tumor phenotype and therefore cannot be considered a proper tumor suppressor. The involvement of VHL and MLH1 also in some specific hereditary cancers allowed to successfully apply linkage analysis for their localization. TGFBR2 might well have a tumor suppressor function. It does reduce tumorigenicity upon transfection. Other 3p genes coding for receptor proteins THRB and RARB, are unlikely candidates for tumor suppression. Present observations on a possible association of FHIT with tumor development leave a number of questions unanswered, so that provisionally it cannot be considered a tumor suppressor. Regions that have been identified as crucial in solid tumor development appear to be at the edge of synteny blocks that have been rearranged through the chromosome evolution which led to the formation of human chromosome 3. Although this may merely represent a chance occurrence, it might also reflect areas of genomic instability.

OBJECTIVE

Age younger than 50 years at the time of colon cancer diagnosis is often used as a screening criterion for Lynch syndrome (hereditary nonpolyposis colorectal cancer syndrome). The purpose of this study was to determine the prevalence of MLH1, MSH2, and MSH6 mutations in an unselected cohort of women diagnosed with endometrial cancer at age younger than 50 years.

METHODS

A prospective, multicenter study was performed at three institutions. After written consent was obtained, germline mutation testing by full sequencing and large deletion analysis of the MLH1, MSH2, and MSH6 genes was performed. Tumor studies included immunohistochemistry of MLH1, MSH2, and MSH6; microsatellite instability analysis; and hypermethylation of the MLH1 promoter.

RESULTS

Of the 100 women, nine (9%; 95% CI, 4.2 to 16.4) carried a deleterious germline mutation: seven women with mutations in MSH2, one woman with a mutation in MLH1, and one woman with a mutation in MSH6. Two additional women had molecular studies consistent with the diagnosis of Lynch syndrome. The mean body mass index (BMI) for the entire cohort was 34.4, which is significantly higher than 29.2, the mean BMI for the mutation carriers. Predictors of finding a germline mutation included having a first-degree relative with a Lynch syndrome-associated cancer, endometrial tumor with loss of MSH2 expression, tumors with high microsatellite instability, and lower BMI.

CONCLUSIONS

In this prospective study of endometrial cancer patients younger than age 50 years, 9% were found to carry germline Lynch syndrome-associated mutations. In addition to young age of onset, family history, BMI, and molecular tumor studies can improve the likelihood of identifying a Lynch syndrome-associated germline mutation in MLH1, MSH2, and MSH6.

We analyzed 921 adenocarcinomas of the esophagus, stomach, colon, and rectum to examine shared and distinguishing molecular characteristics of gastrointestinal tract adenocarcinomas (GIACs). Hypermutated tumors were distinct regardless of cancer type and comprised those enriched for insertions/deletions, representing microsatellite instability cases with epigenetic silencing of MLH1 in the context of CpG island methylator phenotype, plus tumors with elevated single-nucleotide variants associated with mutations in POLE. Tumors with chromosomal instability were diverse, with gastroesophageal adenocarcinomas harboring fragmented genomes associated with genomic doubling and distinct mutational signatures. We identified a group of tumors in the colon and rectum lacking hypermutation and aneuploidy termed genome stable and enriched in DNA hypermethylation and mutations in KRAS, SOX9, and PCBP1.

BACKGROUND

Ductal carcinoma in situ (DCIS) is a non-invasive lesion of the breast that is frequently detected by mammography and subsequently removed by surgery. However, it is estimated that about half of the detected lesions would never have progressed into invasive cancer. Identifying DCIS and invasive cancer specific epigenetic lesions and understanding how these epigenetic changes are involved in triggering tumour progression is important for a better understanding of which lesions are at risk of becoming invasive.

METHODS

Quantitative DNA methylation analysis of ABCB1, CDKN2A/p16INK4a, ESR1, FOXC1, GSTP1, IGF2, MGMT, MLH1, PPP2R2B, PTEN and RASSF1A was performed by pyrosequencing in a series of 27 pure DCIS, 28 small invasive ductal carcinomas (IDCs), 34 IDCs with a DCIS component and 5 normal breast tissue samples. FOXC1, ABCB1, PPP2R2B and PTEN were analyzed in 23 additional normal breast tissue samples. Real-Time PCR expression analysis was performed for FOXC1.

RESULTS

Aberrant DNA methylation was observed in all three diagnosis groups for the following genes: ABCB1, FOXC1, GSTP1, MGMT, MLH1, PPP2R2B, PTEN and RASSF1A. For most of these genes, methylation was already present at the DCIS level with the same frequency as within IDCs. For FOXC1 significant differences in methylation levels were observed between normal breast tissue and invasive tumours (P < 0.001). The average DNA methylation levels were significantly higher in the pure IDCs and IDCs with DCIS compared to pure DCIS (P = 0.007 and P = 0.001, respectively). Real-time PCR analysis of FOXC1 expression from 25 DCIS, 23 IDCs and 28 normal tissue samples showed lower gene expression levels of FOXC1 in both methylated and unmethylated tumours compared to normal tissue (P < 0.001). DNA methylation levels of FOXC1, GSTP1, ABCB1 and RASSF1A were higher in oestrogen receptor (ER) positive vs. ER negative tumours; whereas methylation levels of FOXC1, ABCB1, PPP2R2B and PTEN were lower in tumours with a TP53 mutation.

CONCLUSIONS

Quantitative methylation analysis identified ABCB1, FOXC1, PPP2R2B and PTEN as novel genes to be methylated in DCIS. In particular, FOXC1 showed a significant increase in the methylation frequency in invasive tumours. Low FOXC1 gene expression in both methylated and unmethylated DCIS and IDCs indicates that the loss of its expression is an early event during breast cancer progression.

The WNT/beta-catenin (CTNNB1) pathway is commonly activated in the carcinogenic process. Cross-talks between the WNT and cyclooxygenase-2 (COX-2 or PTGS2)/prostaglandin pathways have been suggested. The relationship between beta-catenin activation and microsatellite instability (MSI) in colorectal cancer has been controversial. The CpG island methylator phenotype (CIMP or CIMP-high) with widespread promoter methylation is a distinct epigenetic phenotype in colorectal cancer, which is associated with MSI-high. However, no study has examined the relationship between beta-catenin activation and CIMP status. Using 832 population-based colorectal cancer specimens, we assessed beta-catenin localization by immunohistochemistry. We quantified DNA methylation in eight CIMP-specific promoters [CACNA1G, CDKN2A(p16), CRABP1, IGF2, MLH1, NEUROG1, RUNX3, and SOCS1] by real-time polymerase chain reaction (MethyLight). MSI-high, CIMP-high, and BRAF mutation were associated inversely with cytoplasmic and nuclear beta-catenin expressions (i.e., beta-catenin activation) and associated positively with membrane expression. The inverse relation between beta-catenin activation and CIMP was independent of MSI. COX-2 overexpression correlated with cytoplasmic beta-catenin expression (even after tumors were stratified by CIMP status), but did not correlate significantly with nuclear or membrane expression. In conclusion, beta-catenin activation is inversely associated with CIMP-high independent of MSI status. Cytoplasmic beta-catenin is associated with COX-2 overexpression, supporting the role of cytoplasmic beta-catenin in stabilizing PTGS2 (COX-2) mRNA.

Twelve to 16% of colorectal cancers (CRCs) display a high degree of microsatellite instability (MSI-H), whereas most are believed to be microsatellite stable (MSS). The existence of a low degree of instability (MSI-L) group has also been proposed. By using the Bethesda panel of microsatellite markers, the microsatellite instability (MSI) status of CRCs can be determined. This set is recommended to distinguish between MSI-H and MSI-L/MSS. No definition for MSI-L has emerged. Most reports on MSI-L rely on the Bethesda panel, using 5-15markers. Tumors with more than 30% MSI are designated as MSI-H, but the lower limit for MSI-L is ambiguous. We hypothesized that if many markers are studied, almost all CRCs would show some MSI. It would be necessary to establish a cutoff level for MSI-L by showing that, above this cutoff level, tumors display molecular and/or clinical features different from those under the cutoff level. To perform this task, we analyzed 90 BAT26 stable CRC samples with 377 markers. MSI at 1-11 loci was observed in 71 (79%) of the 90 cases. K-RAS mutation, loss of heterozygosity, and MLH1 and MGMT hypermethylation analyses were performed, as well as clinical features being scrutinized, to examine possible differences between MSI-L and MSS tumors using all of the possible cutoff levels for MSI-L. Convincing differences between putative MSI-L and MSS groups were not observed. Our results show that the sensitivity of a typically used marker number to detect MSI-L is very low, and they suggest that MSS and MSI-L tumors have a common molecular background.

BACKGROUND

Although up to 30% of patients with colorectal cancer have a positive family history of colorectal neoplasia, few colorectal cancers can be explained by mutations in high-penetrance genes. We investigated whether polymorphisms in DNA mismatch repair genes are associated with the risk of colorectal cancer.

METHODS

We genotyped 929 case patients and 1098 control subjects from Ontario and 430 case patients and 275 control subjects from Newfoundland and Labrador for five polymorphisms in the mismatch repair genes MLH1 and MSH2 with the fluorogenic 5' nuclease assay. Tumor microsatellite instability (MSI) was determined with a polymerase chain reaction-based method; MSI status was assigned as high (MSI-H,>> or = 30% unstable markers among all markers tested), low (MSI-L, <30% markers unstable), or stable (MSS, no unstable markers). We used unconditional logistic regression to evaluate the association between each polymorphism and colorectal cancer after adjusting for age and sex. The associations between polymorphisms and tumor clinicopathologic features were evaluated with a Pearson's chi-square or Fisher's exact test. All statistical tests were two-sided.

RESULTS

We observed strong associations between the MLH1 -93G>A polymorphism and MSI-H tumors among case patients from Ontario (P = .001) and Newfoundland (P = .003). When compared with the control populations, homozygosity for the MLH1 -93G>A variant allele was associated with MSI-H tumors among case patients in Ontario (adjusted odds ratio [OR] = 3.23, 95% confidence interval [CI] = 1.65 to 6.30) and in Newfoundland (OR = 8.88, 95% CI = 2.33 to 33.9), as was heterozygosity among case patients in Ontario (OR = 1.84, 95% CI = 1.20 to 2.83) and in Newfoundland (OR = 2.56, 95% CI = 1.14 to 5.75). Genotype frequencies were similar among case patients with MSS and MSI-L tumors and control subjects, and the majority of homozygous variant carriers had MSS tumors. Among case patients from Ontario, an association between the MLH1 -93G>A polymorphism and a strong family history of colorectal cancer (for Amsterdam criteria I and II, P = .004 and P = .02, respectively) was observed.

CONCLUSIONS

In two patient populations, the MLH1 -93G>A polymorphism was associated with an increased risk of MSI-H colorectal cancer.

The mechanism by which germline mutations of DNA mismatch repair genes cause susceptibility to tumour formation is not yet understood. Studies in vitro indicate that heterozygosity for these mutations, unlike homozygosity, does not affect mismatch repair. Surprisingly, no loss of heterozygosity at the predisposing loci has so far been described in hereditary nonpolyposis colorectal cancers. Here, we show that loss of heterozygosity (LOH) of markers within or adjacent to the MLH1 gene on chromosome 3p occurs nonrandomly in tumours from members of families in which the disease phenotype cosegregates with MLH1. In every informative case, the loss affects the wild type allele. These results suggest that DNA mismatch repair genes resemble tumour suppressor genes in that two hits are required to cause a phenotypic effect.

Hypermethylation of the MLH1 promoter underlies most sporadic colorectal cancers with microsatellite instability (MSI). To investigate the role of hypermethylation in the normal colonic mucosa as a possible precursor lesion, we studied 700 bp upstream of MLH1 covering 51 CpG sites. We found partially methylated alleles in 15 of 34 (44%) patients <60 years of age and 20 of 24 (83%) patients>> or =80 years of age (P = 0.0026). Fully methylated alleles were present in 18 of 33 (55%) patients with MSI+ tumors but in only 18 of 90 (20%) patients with MSI- tumors (P = 0.00019). By in situ analysis, methylation was patchy and located mainly in the cryptal regions close to the lumen. We conclude that the spread of methylation in the MLH1 promoter in the normal colonic mucosa is closely associated with age and the development of sporadic MSI+ colorectal cancers.

The human protein MED1 (also known as MBD4) was previously isolated in a two-hybrid screening using the mismatch repair protein MLH1 as a bait, and shown to have homology to bacterial base excision repair DNA N-glycosylases/lyases. To define the mechanisms of action of MED1, we implemented a sensitive glycosylase assay amenable to kinetic analysis. We show that MED1 functions as a mismatch-specific DNA N-glycosylase active on thymine, uracil, and 5-fluorouracil when these bases are opposite to guanine. MED1 lacks uracil glycosylase activity on single-strand DNA and abasic site lyase activity. The glycosylase activity of MED1 prefers substrates containing a G:T mismatch within methylated or unmethylated CpG sites; since G:T mismatches can originate via deamination of 5-methylcytosine to thymine, MED1 may act as a caretaker of genomic fidelity at CpG sites. A kinetic analysis revealed that MED1 displays a fast first cleavage reaction followed by slower subsequent reactions, resulting in biphasic time course; this is due to the tight binding of MED1 to the abasic site reaction product rather than a consequence of enzyme inactivation. Comparison of kinetic profiles revealed that the MED1 5-methylcytosine binding domain and methylation of the mismatched CpG site are not required for efficient catalysis.

OBJECTIVE

Early-onset colorectal cancer (CRC) represents a clinically distinct form of CRC that is often associated with a poor prognosis. Methylation levels of genomic repeats such as LINE-1 elements have been recognized as independent factors for increased cancer-related mortality. The methylation status of LINE-1 elements in early-onset CRC has not been analyzed previously.

METHODS

We analyzed 343 CRC tissues and 32 normal colonic mucosa samples, including 2 independent cohorts of CRC diagnosed ≤ 50 years old (n=188), a group of sporadic CRC >50 years (MSS n=89; MSI n=46), and a group of Lynch syndrome CRCs (n=20). Tumor mismatch repair protein expression, microsatellite instability status, LINE-1 and MLH1 methylation, somatic BRAF V600E mutation, and germline MUTYH mutations were evaluated.

RESULTS

Mean LINE-1 methylation levels (± SD) in the five study groups were early-onset CRC, 56.6% (8.6); sporadic MSI, 67.1% (5.5); sporadic MSS, 65.1% (6.3); Lynch syndrome, 66.3% (4.5) and normal mucosa, 76.5% (1.5). Early-onset CRC had significantly lower LINE-1 methylation than any other group (p<0.0001). Compared to patients with <65% LINE-1 methylation in tumors, those with ≥ 65% LINE-1 methylation had significantly better overall survival (p=0.026, log rank test).

CONCLUSIONS

LINE-1 hypomethylation constitutes a potentially important feature of early-onset CRC, and suggests a distinct molecular subtype. Further studies are needed to assess the potential of LINE-1 methylation status as a prognostic biomarker for young people with CRC.

Constitutional epimutations of tumor suppressor genes manifest as promoter methylation and transcriptional silencing of a single allele in normal somatic tissues, thereby predisposing to cancer. Constitutional MLH1 epimutations occur in individuals with young-onset cancer and demonstrate non-Mendelian inheritance through their reversal in the germline. We report a cancer-affected family showing dominant transmission of soma-wide highly mosaic MLH1 methylation and transcriptional repression linked to a particular genetic haplotype. The epimutation was erased in spermatozoa but reinstated in the somatic cells of the next generation. The affected haplotype harbored two single nucleotide substitutions in tandem; c.-27C>> A located near the transcription initiation site and c.85G>> T. The c.-27C>> A variant significantly reduced transcriptional activity in reporter assays and is the probable cause of this epimutation.

UNASSIGNED

Germline pathogenic variants in BRCA1 and BRCA2 predispose to an increased lifetime risk of breast cancer. However, the relevance of germline variants in other genes from multigene hereditary cancer testing panels is not well defined.

UNASSIGNED

To determine the risks of breast cancer associated with germline variants in cancer predisposition genes.

UNASSIGNED

A study population of 65 057 patients with breast cancer receiving germline genetic testing of cancer predisposition genes with hereditary cancer multigene panels. Associations between pathogenic variants in non-BRCA1 and non-BRCA2 predisposition genes and breast cancer risk were estimated in a case-control analysis of patients with breast cancer and Exome Aggregation Consortium reference controls. The women underwent testing between March 15, 2012, and June 30, 2016.

UNASSIGNED

Breast cancer risk conferred by pathogenic variants in non-BRCA1 and non-BRCA2 predisposition genes.

UNASSIGNED

The mean (SD) age at diagnosis for the 65 057 women included in the analysis was 48.5 (11.1) years. The frequency of pathogenic variants in 21 panel genes identified in 41 611 consecutively tested white women with breast cancer was estimated at 10.2%. After exclusion of BRCA1, BRCA2, and syndromic breast cancer genes (CDH1, PTEN, and TP53), observed pathogenic variants in 5 of 16 genes were associated with high or moderately increased risks of breast cancer: ATM (OR, 2.78; 95% CI, 2.22-3.62), BARD1 (OR, 2.16; 95% CI, 1.31-3.63), CHEK2 (OR, 1.48; 95% CI, 1.31-1.67), PALB2 (OR, 7.46; 95% CI, 5.12-11.19), and RAD51D (OR, 3.07; 95% CI, 1.21-7.88). Conversely, variants in the BRIP1 and RAD51C ovarian cancer risk genes; the MRE11A, RAD50, and NBN MRN complex genes; the MLH1 and PMS2 mismatch repair genes; and NF1 were not associated with increased risks of breast cancer.

UNASSIGNED

This study establishes several panel genes as high- and moderate-risk breast cancer genes and provides estimates of breast cancer risk associated with pathogenic variants in these genes among individuals qualifying for clinical genetic testing.

Colon cancer is the third leading cause of cancer-related death in the United States, affecting approximately 147,000 people each year. Most colon cancers arise from benign neoplasms and evolve into adenocarcinomas through a stepwise histologic progression sequence that starts from adenomas or hyperplastic polyps/serrated adenomas. Genetic alterations and, more recently, epigenetic alterations have been associated with specific steps in this polyp-adenocarcinoma sequence and likely drive the histologic progression of colon cancer. Consequently, we have assessed in colon adenomas and hyperplastic polyps the methylation status of MGMT, CDKN2A, and MLH1 to determine the timing and frequency of these events in the polyp-carcinoma progression sequence and subsequently to analyze the potential for these methylated genes to be molecular markers for adenomas and hyperplastic polyps. We have found that methylated MGMT, CDKN2A, and MLH1 occur in 49%, 34%, and 7% of adenomas and in 5%, 10%, and 7% of hyperplastic polyps, respectively, and that they are more common in histologically advanced adenomas. Furthermore, analysis of fecal DNA from persons who have undergone colonoscopic exams revealed methylated CDKN2A, MGMT, and MLH1 in fecal DNA from 31%, 48%, and 0% of individuals with adenomas and from 16%, 27%, and 10% of individuals with no detectable polyps, respectively. These results show that aberrant methylated genes can be detected frequently in sporadic colon polyps and that they can be detected in fecal DNA. Notably, improvements in the specificity and sensitivity of the fecal DNA-based assays will be needed to make them clinically useful diagnostic tests for polyps.

BACKGROUND

Multiple epigenetic and genetic changes have been reported in colorectal tumors, but few of these have clinical impact. This study aims to pinpoint epigenetic markers that can discriminate between non-malignant and malignant tissue from the large bowel, i.e. markers with diagnostic potential. The methylation status of eleven genes (ADAMTS1, CDKN2A, CRABP1, HOXA9, MAL, MGMT, MLH1, NR3C1, PTEN, RUNX3, and SCGB3A1) was determined in 154 tissue samples including normal mucosa, adenomas, and carcinomas of the colorectum. The gene-specific and widespread methylation status among the carcinomas was related to patient gender and age, and microsatellite instability status. Possible CIMP tumors were identified by comparing the methylation profile with microsatellite instability (MSI), BRAF-, KRAS-, and TP53 mutation status.

RESULTS

The mean number of methylated genes per sample was 0.4 in normal colon mucosa from tumor-free individuals, 1.2 in mucosa from cancerous bowels, 2.2 in adenomas, and 3.9 in carcinomas. Widespread methylation was found in both adenomas and carcinomas. The promoters of ADAMTS1, MAL, and MGMT were frequently methylated in benign samples as well as in malignant tumors, independent of microsatellite instability. In contrast, normal mucosa samples taken from bowels without tumor were rarely methylated for the same genes. Hypermethylated CRABP1, MLH1, NR3C1, RUNX3, and SCGB3A1 were shown to be identifiers of carcinomas with microsatellite instability. In agreement with the CIMP concept, MSI and mutated BRAF were associated with samples harboring hypermethylation of several target genes.

CONCLUSIONS

Methylated ADAMTS1, MGMT, and MAL are suitable as markers for early tumor detection.