Women with germ-line mutations in the mismatch repair genes (responsible for hereditary nonpolyposis colorectal cancer) face an increased risk of colonic and endometrial cancer. However, these germ-line mutations are rare and are responsible for fewer than 1% of endometrial cancers. Therefore, we examined whether or not common variants of the hereditary nonpolyposis colorectal cancer-associated genes might also be associated with an increased risk of endometrial cancer. Three single-nucleotide polymorphisms were selected in the MLH1 and MSH2 mismatch repair genes. All the various 672 women with endometrial cancer and 880 controls were genotyped. Each of these three single-nucleotide polymorphisms was associated with an increased risk of endometrial cancer. Carriers of the MLH1 nt-93 A allele were at a 1.5-fold increased risk of developing endometrial cancer compared with controls [95% confidence interval (95% CI), 1.2-2.0; P = 0.001]. The risk was higher for homozygote carriers [odds ratio (OR), 1.9; 95% CI, 1.2-3.2; P = 0.009]. For carriers of the MSH2 rs2303428 C allele, the OR was 1.4 (95% CI, 1.0-1.9; P = 0.05), and for carriers of the MSH2 rs2059520 G allele, the OR was 1.3 (95% CI, 1.0-1.7; P = 0.03). More than 9% of endometrial cancer cases carried a variant allele in both MLH1 and MSH2. For these women, the risk of endometrial cancer was particularly high (OR, 2.1; 95% CI, 1.2-3.6; P = 0.005). For patients younger than 50 years at diagnosis who carried both variants, the risk was even higher (OR, 3.4; 95% CI, 1.7-6.6; P = 0.0005). In summary, two common variant alleles of the MLH1 and MSH2 genes make a substantial contribution to endometrial cancer incidence in Ontario.

Nonrandom, widespread promoter methylation of tumor suppressor genes is a common mechanism of gene inactivation during tumorigenesis. We examined the methylation status of two distinct regions of the MLH1 promoter (proximal and distal to the transcription start site) and the MLH1 gene expression by methylation-specific PCR and immunohistochemistry. A total of 72 colorectal tumors, both with (n = 51, 22 affected by hereditary nonpolyposis colorectal cancer, HNPCC, defined according to the international clinical criteria and 29 sporadic cases) and without microsatellite instability (MSI) (n = 21) were studied. Methylation was present in at least one of the two promoter regions in 86% of the sporadic MSI cases, in 33% of the cases lacking MSI, and in 23% of the HNPCC tumors. In the HNPCC cases with a known MLH1 mutation (n = 10) none of the two promoter regions was methylated. Hypermethylation in both MLH1 promoter regions was seen in 45% of the MSI sporadic cases vs. 5% of the MSI-negative cases and 0% of the HNPCC cases. The overall concordance between the two promoter regions regarding methylation status was good (P = 0.009), but no significant correlation between methylation and suppression of the MLH1 immunohistochemical expression was found. Our data confirm that mutation and hypermethylation are mutually exclusive mechanisms in inducing mismatch repair deficiency and support the hypothesis of methylation as a process evenly distributed along the different regions of the promoter.

HNPCC is an autosomal dominantly inherited cancer-susceptibility syndrome that confers an increased risk for colorectal cancer and endometrial cancer at a young age. It also entails an increased risk of a variety of other tumors, such as ovarian, gastric, uroepithelial and biliary tract cancers. The underlying pathogenic mutation lies in 1 of the 5 known DNA MMR genes (MSH2, MLH1, PMS1, PMS2 and MSH6). We screened a total of 140 individuals from 56 Spanish families with suspected HNPCC for mutations in the DNA mismatch repair genes MLH1 and MSH2, using DGGE and direct DNA sequencing. Families were selected on the basis of a history of HNPCC-related tumors or the occurrence of other associated tumors in members besides the index case affected with colorectal cancer. We detected 14 definite pathogenic germline mutations, 9 in MLH1 and 5 in MSH2 in 13 unrelated families selected by the Amsterdam criteria and Bethesda guidelines (1 family carries 2 mutations) and 3 missense mutations in 3 unrelated families selected by the Amsterdam criteria. Among the 17 germline mutations noted in the Spanish cohort, 10 are novel, 7 in MLH1 and 3 in MSH2, perhaps demonstrating different mutational spectra in the Spanish population, where no founder mutation has been identified. Based on our results, we suggest that in the Spanish population not only HNPCC families fulfilling the Amsterdam criteria but also those following Bethesda guidelines should undergo genetic testing for MSH2 and MLH1 mutations.

PolyA simple repeat sequence deletions are common in tumors with microsatellite instability (MSI+). Such deletions occur one base at a time in DNA mismatch repair (MMR)-deficient yeast suggesting larger deletions in human MSI+ tumors represent multiple sequential stepwise losses. Sum total deletions in four polyA repeats were variable (between -17 to -45 bp) in 20 sporadic MSI+ colorectal cancers. Progressive but less extensive total deletions (maximum of -12 bp) occurred in similar polyA sequences in MMR-deficient mice (mlh1-/-) up to 478 days old. PolyA repeat lengths were relatively stable but already shortened in the MMR-deficient cell line HCT116. A transgene with 26 A's transfected into HCT116 shortened an average of 3.8 bases pairs after 469 days in culture, less than average deletions of BAT25 (-5.3) or BAT26 (-9.0) in MSI+ cancers. These findings further suggest that extensive polyA deletions common in MSI+ tumors likely reflect multiple stepwise smaller deletions that accumulate more than hundreds of divisions after loss of MMR.

BACKGROUND

Cancer-specific hypermethylation of (promoter) CpG islands is common during the tumorigenesis of colon cancer. Although associations between certain genetic aberrations, such as BRAF mutation and microsatellite instability, and the CpG island methylator phenotype (CIMP), have been found, the mechanisms by which these associations are established are still unclear. We studied genome-wide DNA methylation differences between colorectal tumors carrying a BRAF mutation and BRAF wildtype tumors.

RESULTS

Using differential methylation hybridization on oligonucleotide microarrays representing 32,171 CpG-rich regions, we identified 1,770 regions with differential methylation between colorectal tumor and paired normal colon. Next, we compared the tumor/normal methylation ratios between different groups of patients. Related to CIMP, we identified 749 differentially methylated regions, of which 86% had a higher tumor/normal methylation ratio in the CIMP-positive group. We identified 758 regions with a BRAF mutation-specific methylation change, of which 96% had a higher tumor/normal methylation ratio in the BRAF mutant group. Among the genes affected by BRAF mutation-specific methylation changes, we found enrichment of several cancer-related pathways, including the PI3 kinase and Wnt signaling pathways. To focus on genes that are silenced in a tumor-specific rather than a lineage-specific manner, we used information on the epigenetic silencing mark H3K27me3 in embryonic stem (ES) cells. Among the genes showing BRAF mutation-specific promoter methylation but no H3K27me3 mark in ES cells were forkhead box (FOX) transcription factors associated with the PI3 kinase pathway, as well as MLH1 and SMO. Repression of FOXD3 gene expression in tumors could be related to its promoter hypermethylation.

CONCLUSIONS

We identified new BRAF mutation-specific methylation changes in colorectal cancer. Epigenetic downregulation of these targets may contribute to mutationally active BRAF-driven tumorigenesis, explaining its association with aberrant DNA methylation.

Gemcitabine is a cytotoxic cytidine analog, which is widely used in anti-cancer therapy. One mechanism by which gemcitabine acts is by inhibiting nucleotide excision repair (NER). Recently NER was implicated in Gadd45 mediated DNA demethylation and epigenetic gene activation. Here we analyzed the effect of gemcitabine on DNA demethylation. We find that gemcitabine inhibits specifically Gadd45a mediated reporter gene activation and DNA demethylation, similar to the topoisomerase I inhibitor camptothecin, which also inhibits NER. In contrast, base excision repair inhibitors had no effect on DNA demethylation. In Xenopus oocytes, gemcitabine inhibits DNA repair synthesis accompanying demethylation of oct4. In mammalian cells, gemcitabine induces DNA hypermethylation and silencing of MLH1. The results indicate that gemcitabine induces epigenetic gene silencing by inhibiting repair mediated DNA demethylation. Thus, gemcitabine can function epigenetically and provides a tool to manipulate DNA methylation.

To compare protein expression levels, gene mutation and survival among Right-Sided Colon Cancer (RSCC), Left-Sided Colon Cancer (LSCC) and rectal cancer patients, 57 cases of RSCC, 87 LSCC and 145 rectal cancer patients were included retrospectively. Our results demonstrated significant differences existed among RSCC, LSCC and rectal cancer regarding tumor diameter, differentiation, invasion depth and TNM stage. No significant difference was identified in expression levels of MLH1, MSH2, MSH6, PMS2, β-Tubulin III, P53, Ki67 and TOPIIα, and gene mutation of KRAS and BRAF among three groups. Progression Free Survival (PFS) of RSCC was significantly lower than that of LRCC and rectal cancer. In univariate analyses, RSCC, preoperative chemoradiotherapy, poor differentiation, advanced TNM stage, elevated serum CEA and CA19-9 level, tumor deposit, perineural and vascular invasion were found to be predictive factors of shorter PFS. In multivariate analyses, only differentiation and TNM stages were found to be independent predictors of PFS. In conclusion, compared with LSCC and rectal cancer, RSCC has larger tumor size, poor differentiation, advanced TNM stage and shorter survival. The shorter survival in RSCC might be attributed to the advanced tumor stage caused by its inherent position feature of proximal colon rather than genetic difference.

In the zebra finch (Taeniopygia guttata), there is a germ-line-restricted chromosome regularly present in males and females. A reexamination of male and female meiosis in the zebra finch showed that this element forms a euchromatic bivalent in oocytes, but it is always a single, heterochromatic element in spermatocytes. Immunostaining with anti-MLH1 showed that the bivalent in oocytes has two or three foci with a localized pattern, indicating the regular occurrence of recombination. In male meiosis, the single restricted chromosome forms an axis that contains the cohesin subunit SMC3, and the associated chromatin is densely packed until late pachytene. Electron microscopy of thin-sectioned seminiferous tubules shows that the restricted chromosome is eliminated in postmeiotic stages in the form of packed chromatin inside a micronucleus, visible in the cytoplasm of young spermatids. The selective condensation of the restricted chromosome during early meiotic prophase in males is interpreted as a strategy to avoid the triggering of asynaptic checkpoints, but this condensation is reversed prior to the final condensation that leads to its (ulterior) elimination. Recombination during female meiosis may prevent the genetic attrition of the restricted chromosome and, along with the elimination in male germ cells, ensures its regular transmission through females.

DNA mismatch repair (MMR) is involved in the post-replication correction of errors due to misincorporated nucleotides or DNA slippage during DNA synthesis. We previously reported the reduction or loss of MMR protein expression in human prostate cancer cell lines and some primary tumors. In the present report, we further demonstrate the involvement of defects of MMR in the pathogenesis of prostate cancer. Immunohistochemical analysis of 39 formalin-fixed, paraffin-embedded human prostate tumors, showed reduction or absence of MMR protein expression (MLH1, MSH2, PMS2) in the epithelium of prostate tumor foci compared to normal adjacent prostate tissue. The reduction or absence of the PMS2 and MSH2 (but not MLH1) protein was correlated to the differentiation of the tumor. Poorly differentiated tumors showed greater loss of these two proteins than the well differentiated tumors (P<0.05). We previously reported that microsatellite instability was detectable by a beta-galactosidase restoration mutation assay in the prostate cancer cell lines DU145, PC3, LNCaP, p67SV40T, M2182, and M12. In this study, we detected the insertion or deletion of one nucleotide in the mononucleotide repeats located within the coding regions of BAX gene in DU145, and TGFbetaRII in M12 cells. In addition, we used an in vitro model of defective MMR to demonstrate that microsatellite instability can be induced in an otherwise stable cancer cell line by transfection with a dominant negative fragment of PMS2. These results suggest that defects in MMR may result in MSI in the secondary genes in prostate cancer. From these results, we conclude that loss of MMR function can produce MSI and target some secondary genes containing microsatellites in their coding regions. These series of events may play important roles in the development of human prostate cancer.

Mutations in the mismatch repair (MMR) genes hMSH2 and hMLH1 have been associated with hereditary nonpolyposis colorectal cancer. Tumor cell lines that are deficient in MMR exhibit a high mutation rate, a defect in the response to certain types of DNA damage and in transcription-coupled repair, as well as an increase in the rate of gene amplification. We show here that hMSH2-deficient tumor cell lines lost most of their ability to accurately repair plasmid DNA double-strand breaks (DSBs) by homologous recombination, compared with MMR-proficient or hMLH1-deficient tumor cell lines. In all of these cell lines, DSB repair occurred almost exclusively by nonreciprocal homologous recombination: gene conversion (GC). However, there were two types of GC products: precise and rearranged. The rearranged products contained deletions or insertions of sequences and represented GC intermediates trapped at various stages and shunted to nonhomologous end joining. In MMR-proficient or MLH1-deficient cells, >50% of GC products were of the precise type, whereas in two MSH2-deficient backgrounds, this proportion decreased to 8%, whereas that of rearranged GC products increased by 2-fold. These results seem to predict a novel way by which MSH2-deficiency could promote mutation: deletion or insertion mutations associated with DSB repair, which may also contribute to cancer predisposition.

Women with Lynch syndrome have an additional need to address the substantial increased lifetime risk of endometrial and ovarian cancer. Endometrial or ovarian cancer can be the presenting cancer in individuals with Lynch syndrome, or can be a second cancer. Lifetime risk of endometrial cancer in women with MLH1 or MSH2 mutations is approximately 40 %, with a median age of 49. Women with MSH6 mutations have a similar risk of endometrial cancer but a later age of diagnosis. Lynch syndrome-associated endometrial cancers are primarily endometrioid, although non-endometrioid subtypes including clear cell, papillary serous and MMMT have been reported. In addition, endometrial cancers arising in the lower uterine segment, while rare in the general population, are enriched in women with Lynch syndrome. Ovarian cancer risk in women with Lynch syndrome is 6-8 %, and Lynch syndrome-associated ovarian cancers exhibit a variety of histopathological subtypes. Studies of endometrial cancer screening in Lynch syndrome have been small, and more recently have focused on the use of office endometrial biopsy to identify pre-malignant and early stage cancers. Prevention options include the use of oral contraceptives, which are known to be highly effective for decreasing risk of both endometrial and ovarian cancer in the general population, and prophylactic surgery to remove the uterus and ovaries.

We report the molecular characterization of 8 primary gastric carcinomas, corresponding xenografts, and 2 novel gastric carcinoma cell lines. We compared the tumors and cell lines, with respect to histology, immunohistochemistry, copy number, and hypermethylation of up to 38 genes using methylation-specific multiplex ligation-dependent probe amplification, and TP53 and CDH1 mutation analysis where relevant. The primary tumors and xenografts were histologically comparable and shared expression of 11 of 14 immunohistochemical markers (E-cadherin, beta-catenin, COX-2, p53, p16, TFF1, cyclin E, MLH1, SMAD4, p27, KLK3, CASR, CHFR, and DAPK1). Gains of CASR, DAPK1, and KLK3--not yet described in gastric cancer--were present in the primary tumors, xenografts, and cell lines. The most prominent losses occurred at CDKN2A (p16), CDKN2B (p15), CDKN1B (p27/KIP1), and ATM. Except for ATM, these losses were found only in the cell line or xenograft, suggesting an association with tumor progression. However, examination of p16 and p27 in 174 gastric cancers using tissue microarrays revealed no significant correlation with tumor stage or lymph node status. Further losses and hypermethylation were detected for MLH1, CHFR, RASSF1, and ESR, and were also seen in primary tumors. Loss of CHFR expression correlated significantly with the diffuse phenotype. Interestingly, we found the highest rate of methylation in primary tumors which gave rise to cell lines. In addition, both cell lines harbored mutations in CDH1, encoding E-cadherin. Xenografts and gastric cancer cell lines remain an invaluable research tool in the uncovering of the multistep progression of cancer. The frequent gains, losses, and hypermethylation reported in this study indicate that the involved genes or chromosomal regions may be relevant to gastric carcinogenesis.

OBJECTIVE

Lynch syndrome (LS) patients have DNA mismatch repair deficiency and up to 80% lifetime risk of colorectal cancer (CRC). Screening of mutation carriers reduces CRC incidence and mortality. Selection for constitutional mutation testing relies on family history (Amsterdam and Bethesda Guidelines) and tumour-derived biomarkers. Initial biomarker analysis uses mismatch repair protein immunohistochemistry and microsatellite instability. Abnormalities in either identify mismatch repair deficiency but do not differentiate sporadic epigenetic defects, due to MLH1 promoter region methylation (13% of CRCs) from LS (4% of CRCs). A diagnostic biomarker capable of making this distinction would be valuable. This study compared two biomarkers in tumours with mismatch repair deficiency; quantification of methylation of the MLH1 promoter region using a novel assay and BRAF c.1799T>A, p.(Val600Glu) mutation status in the identification of constitutional mutations.

METHODS

Tumour DNA was extracted (formalin fixed, paraffin embedded, FFPE tissue) and pyrosequencing used to test for MLH1 promoter methylation and presence of the BRAF c.1799T>A, p.(Val600Glu) mutation 71 CRCs from individuals with pathogenic MLH1 mutations and 73 CRCs with sporadic MLH1 loss. Specificity and sensitivity was compared.

UNASSIGNED

Unmethylated MLH1 promoter: sensitivity 94.4% (95% CI 86.2% to 98.4%), specificity 87.7% (95% CI 77.9% to 94.2%), Wild-type BRAF (codon 600): sensitivity 65.8% (95% CI 53.7% to 76.5%), specificity 98.6% (95% CI 92.4% to 100.0%) for the identification of those with pathogenic MLH1 mutations.

CONCLUSIONS

Quantitative MLH1 promoter region methylation using pyrosequencing is superior to BRAF codon 600 mutation status in identifying constitutional mutations in mismatch repair deficient tumours.

The spectrum of cancers seen in a hospital based Lynch syndrome registry of mismatch repair gene mutation carriers was examined to determine the distribution of cancers and examine excess cancer risk. Overall there were 504 cancers recorded in 368 mutation carriers from 176 families. These included 236 (46.8 %) colorectal and 268 (53.2 %) extracolonic cancers. MLH1 mutation carriers had a higher frequency of colorectal cancers whereas MSH2, MSH6 and PMS2 mutation carriers had more extracolonic cancers although these differences were not statistically significant. Men had fewer extracolonic cancers than colorectal (45.3 vs. 54.7 %), whereas women had more extracolonic than colorectal cancers (59.0 vs. 41.0 %). The mean age at diagnosis overall for extracolonic cancers was older than for colorectal, 49.1 versus 44.8 years (P ≤ 0.001). As expected, the index cancer was colorectal in 58.1 % of patients and among the extracolonic index cancers, endometrial was the most common (13.8 %). A significant number of non-Lynch syndrome index cancers were recorded including breast (n = 5) prostate (n = 3), thyroid (n = 3), cervix (n = 3), melanoma (n = 3), and 1 case each of thymoma, sinus cavity, and adenocarcinoma of the lung. However, standardized incidence ratios calculated to assess excess cancer risk showed that only those cancers known to be associated with Lynch syndrome were significant in our sample. We found that Lynch syndrome patients can often present with cancers that are not considered part of Lynch syndrome. This has clinical relevance both for diagnosis of Lynch syndrome and surveillance for cancers of different sites during follow-up of these patients.

BACKGROUND

Platinum-based chemotherapy is besides the standard antifolate therapy with pemetrexed, the cornerstone for treatment of patients with malignant pleural mesothelioma (MPM), and its efficacy depends on several DNA repair enzymes. Therefore, these enzymes could be biomarkers for "tailoring" chemotherapy. This study evaluated enzymes involved in repair of platinum-caused DNA damage, potentially resulting in a biomarker panel associated with patient response and outcome to platinum-based chemotherapy.

METHODS

Pre- or posttreatment specimens from a total of 103 patients with MPM who were undergoing first-line chemotherapy were tested separately. Immunohistochemistry for ERCC1 (endonuclease excision repair cross-complementing 1), MLH1 (MutL homologue 1), MutS homologue (MSH) 2, MSH6, and βIII-tubulin protein expression, and pyrosequencing for ERCC1 codon 118 and C8092A polymorphisms were performed, and their results were correlated to clinicopathologic data.

RESULTS

ERCC1, MLH1, MSH2, MSH6, and βIII-tubulin were expressed in human MPM specimens at different intensities. When considering only pretreatment specimens, MSH6 protein levels were correlated to progression during chemotherapy (P = .0281). MLH1 protein levels (P = .0205), and ERCC1 codon 118 polymorphisms (P ≤ .0001) were significantly associated with progression-free survival. A significant association between ERCC1 protein levels and overall survival was noted (P = .032). Analyses of posttreatment specimens revealed significant associations between βIII-tubulin protein levels and progression-free survival (P = .0066). ERCC1 C8092A polymorphisms were significantly associated with progression-free survival and overall survival (P = .0463 and P = .0080, respectively) in this group.

CONCLUSIONS

Enzymes involved in DNA repair mechanisms are associated with patient response and outcome to platinum-based chemotherapy. Their assessment may be a helpful tool to tailor platinum-based chemotherapy of MPM patients who might expect the largest clinical benefit. Prospective validation of this biomarker panel is warranted.

OBJECTIVE

To determine whether DNA mismatch repair (MMR) modifies the response to chemotherapy or radiotherapy in patients with endometrial cancer.

METHODS

Immunohistochemistry (IHC) for the DNA MMR proteins MLH1, MSH2, MSH6, and PMS2 was performed on a tissue microarray of specimens of primary endometrial cancer. MMR deficiency was defined as lack of expression of one or more proteins. Expression of all proteins classified a tumor as having an intact MMR system. Recurrence rates were calculated for women treated with platinum-based chemotherapy or pelvic external beam radiation. Comparisons were made using the log-rank test. Multiple comparisons were controlled for by utilizing the Bonferroni correction method.

RESULTS

Four hundred seventy-seven cases of endometrial cancer were evaluated on a tissue microarray (TMA). One hundred fifty-eight patients (41%) received chemotherapy. Sixty-six patients (17%) received pelvic teletherapy. Overall and progression-free survival were not different between patients whose tumors had intact MMR and those with defective MMR when stratified by adjuvant treatment with radiation or chemotherapy. Subgroup analyses stratified by histology (non-endometrioid versus endometrioid) and stage did show significant survival differences. There was a significant increase in overall (p=0.003) and progression-free (p=0.004) survival in those with MMR-deficient, non-endometrioid tumors treated with teletherapy compared to those with an intact MMR system. Improved progression-free survival was noted in patients with intact MMR with stage III/IV disease treated with adjuvant chemotherapy (p=0.031).

CONCLUSIONS

Subgroups of patients with non-endometrioid endometrial cancer and defective MMR may have improved survival after adjuvant radiotherapy. Patients with advanced stage endometrial cancer and defects in mismatch repair may receive less benefit from adjuvant chemotherapy.

The DNA damage response (DDR) pathway coordinates the identification, signaling, and repair of DNA damage caused by endogenous or exogenous factors and regulates cell-cycle progression with DNA repair to minimize DNA damage being permanently passed through cell division. Severe DNA damage that cannot be repaired may trigger apoptosis; as such, the DDR pathway is of crucial importance as a cancer target. Poly (ADP-ribose) polymerase (PARP) is the best-known element of the DDR, and several PARP inhibitors have been licensed. However, there are approximately 450 proteins involved in DDR, and a number of these other targets are being investigated in the laboratory and clinic. We review the most recent evidence for the clinical effect of PARP inhibition in breast and ovarian cancer and explore expansion into the first-line setting and into other tumor types. We critique the evidence for patient selection techniques and summarize what is known about mechanisms of PARP inhibitor resistance. We then discuss what is known about the preclinical rationale for targeting other members of the DDR pathway and the associated tumor cell genetics that may confer sensitivity to these agents. Examples include DNA damage sensors (MLH1), damage signaling molecules (ataxia-telangiectasia mutated; ataxia-telangiectasia mutated-related and Rad3-related; CHK1/2; DNA-dependent protein kinase, catalytic subunit; WEE1; CDC7), or effector proteins for repair (POLQ [also referred to as POLθ], RAD51, poly [ADP-ribose] glycohydrolase). Early-phase clinical trials targeting some of these molecules, either as a single agent or in combination, are discussed. Finally, we outline the challenges that must be addressed to maximize the therapeutic opportunity that targeting DDR provides.

Ovarian malignancies occurring in the setting of hereditary nonpolyposis colorectal carcinoma syndrome typically present in young women, often as the first or "sentinel" cancer, but the frequency of microsatellite instability (MSI) and mismatch repair (MMR) defects in ovarian surface epithelial malignancies in women <or=50 years of age is neither well known nor well tested. Fifty-two ovarian surface epithelial carcinomas, including 4 with synchronous endometrial carcinomas, were identified in patients 50 years of age or younger and evaluated for evidence of MSI and MMR protein deficiency. Each case was tested for MSI by multiplex polymerase chain reaction amplification of the National Cancer Institute reference panel (BAT25, BAT26, D2S123, D5S346, and D17S250) and deficiency of MMR protein expression by immunohistochemistry (<em>MLH1</em>, MSH2, MSH6, and PMS2). MMR protein expression and MSI (in a subset of cases) were also evaluated in 50 unselected ovarian serous tumors of low malignant potential , a tumor common in younger women. Defects in MMR were detected in 5 of 52 (10%) ovarian carcinomas by at least 1 testing method, including 2 of 4 (50%) ovarian cancers presenting with synchronous endometrial cancer. Three of the 5 (60%) ovarian carcinomas were clear cell carcinomas (17% of all pure clear cell carcinomas) and the remaining 2 were high-grade carcinomas with endometrioid and mixed histology. Loss of MSH2 and MSH6 was detected in all of the affected clear cell carcinomas and a synchronous endometrial cancer with endometrioid histology. Loss of 1 or more MMR proteins was initially noted in 10/50 ovarian serous tumors of low malignant potential on tissue microarray, but further testing on full tissue sections showed intact protein expression and microsatellite stability in all informative cases. This study demonstrates a 10% rate of MMR-deficient ovarian cancer in women <or=50 years of age. MMR-deficient ovarian cancer is frequently associated with loss of expression of MSH2 and MSH6 proteins and clear cell histology. The occurrence of MMR inactivation in a significant proportion of ovarian clear cell carcinomas (17% in this study) suggests that this tumor may warrant targeted testing in women <or=50 years of age.

BACKGROUND

Microsatellite-unstable colorectal cancers (CRC) that are due to deficient DNA mismatch repair (dMMR) represent approximately 15% of all CRCs in the United States. These microsatellite-unstable CRCs represent a heterogenous group of diseases with distinct oncogenesis pathways. There are overlapping clinicopathologic features between some of these groups, but many important differences are present. Therefore, determination of the etiology for the dMMR is vital for proper patient management and follow-up.

UNASSIGNED

Epigenetic inactivation of MLH1 MMR gene (sporadic microsatellite-unstable CRC) and germline mutation in an MMR gene (Lynch syndrome, LS) are the two most common mechanisms in the pathogenesis of microsatellite instability in CRC. However, in a subset of dMMR CRC cases that are identified by screening tests, no known LS-associated genetic alterations are appreciated by current genetic analysis. When the etiology for dMMR is unclear, it leads to patient anxiety and creates challenges for clinical management.

CONCLUSIONS

It is critical to distinguish LS patients from other patients with tumors due to dMMR, so that the proper screening protocol can be employed for the patients and their families, with the goal to save lives while avoiding unnecessary anxiety and costs. This review summarizes the major pathogenesis pathways of dMMR CRCs, their clinicopathologic features, and practical screening suggestions. In addition, we include frequently asked questions for MMR immunohistochemistry interpretation.

BACKGROUND

The ability to maintain DNA integrity is a critical cellular function. DNA repair is conducted by distinct pathways of genes, many of which are thought to be altered in colorectal cancer. However, there has been little characterization of these pathways in colorectal cancer.

METHODS

By using the TaqMan real-time quantitative PCR, RNA expression profiling of 20 DNA repair pathway genes was done in matched tumor and normal tissues from 52 patients with Dukes' C colorectal cancer.

RESULTS

The relative mRNA expression level across the 20 DNA repair pathway genes varied considerably, and the individual variability was also quite large, with an 85.4 median fold change in the tumor tissue genes and a 127.2 median fold change in the normal tissue genes. Tumor-normal differential expression was found in 13 of 20 DNA repair pathway genes (only XPA had a lower RNA level in the tumor samples; the other 12 genes had significantly higher tumor levels, all P<0.01). Coordinated expression of ERCC6, HMG1, MSH2, and POLB (RS>or=0.60) was observed in the tumor tissues (all P<0.001). Apoptosis index was not correlated with expression of the 20 DNA repair pathway genes. MLH1 and XRCC1 RNA expression was correlated with microsatellite instability status (P=0.045 and 0.020, respectively). An inverse correlation was found between tumor MLH1 RNA expression and MLH1 DNA methylation (P=0.003).

CONCLUSIONS

Our study provides an initial characterization of the DNA repair pathways for understanding the cellular DNA damage/repair system in human colorectal cancer.

Transcriptional inactivation of tumor-suppressor genes by promoter CpG island methylation is thought to be an important mechanism in human carcinogenesis. The CpG island methylator phenotype (CIMP) with extensive promoter methylation appears to be a distinct epigenetic subtype of colorectal carcinoma. Most previous studies on CpG island methylation in colorectal carcinoma used methylation-specific PCR, which may detect low levels of DNA methylation with little or no biological significance. In contrast, quantitative DNA methylation assays have been shown to provide useful information beyond that which can be achieved with methylation-specific PCR. Synchronous neoplasias provide a unique model for investigators to examine molecular alterations in multistep tumorigenesis within one individual. However, no study to date has quantified DNA methylation of CIMP-specific promoters in synchronous colorectal neoplasias. Utilizing real-time PCR (MethyLight), we quantified DNA methylation in five CIMP-specific gene promoters [CACNA1G (calcium channel, voltage-dependent, T type alpha-1G subunit), CDKN2A (p16/INK4A), CRABP1 (cellular retinoic acid binding protein-1), MLH1 and NEUROG1 (neurogenin 1)] and MGMT in six synchronous carcinoma pairs (12 carcinomas) and eight synchronous carcinoma and adenoma pairs (16 tumors). We found that while some synchronous tumor pairs showed discordant promoter methylation patterns, other tumor pairs showed similar, but not exactly identical, patterns of promoter methylation. All but two pairs showed concordant patterns of CIMP status (CIMP positive vs CIMP negative) (P = 0.05 in cancer pairs). BRAF mutations were present in only CIMP-positive tumors. A high degree of microsatellite instability (MSI-H) was observed in both CIMP-positive and CIMP-negative tumors. KRAS mutations were not concordant in any synchronous neoplasia pair. In conclusion, epigenetic alterations at CIMP-specific promoter CpG islands in synchronous colorectal neoplasias likely have both random and nonrandom components.

OBJECTIVE

We have performed a case-control study among prostate cancer patients treated with three-dimensional conformational radiotherapy (3D-CRT) in order to investigate the association between single nucleotide polymorphisms (SNPs), treatment and patient features with gastrointestinal and genitourinary acute toxicity.

METHODS

A total of 698 patients were screened for 14 SNPs located in the ATM, ERCC2, LIG4, MLH1 and XRCC3 genes. Gastrointestinal and genitourinary toxicities were recorded prospectively using the Common Terminology Criteria for Adverse Events v3.0.

RESULTS

The XRCC3 SNP rs1799794 (G/G OR=5.65; 95% CI: 1.95-16.38; G/A OR=2.75; 95% CI: 1.25-6.05; uncorrected p-value=2.8×10(-03); corrected p-value=0.03; FDR q-value=0.06) as well as the mean dose received by the rectum (OR=1.06; 95% CI: 1.02-1.1; uncorrected p-value=2.49×10(-03); corrected p-value=0.03; FDR q-value=0.06) were significantly associated with gastrointestinal toxicity after correction for multiple testing. Those patients who undergone previous prostatectomy were less prone to develop genitourinary toxicity (OR=0.38; 95% CI: 0.18-0.71; uncorrected p-value=4.95×10(-03); corrected p-value=0.03; FDR q-value=0.08). Our study excludes the possibility of a >2-fold risk increase in genitourinary acute toxicity being due to rs1801516 ATM SNP, the rs1805386 and rs1805388 LIG4 markers, as well as all the SNPs evaluated in the ERCC2, MLH1 and XRCC3 genes.

CONCLUSIONS

The XRCC3 rs1799794 SNP and the mean dose received by the rectum are associated with the development of gastrointestinal toxicity after 3D-CRT.

To investigate the relationship between fenofibrate (FF) and oxidative stress, enzymatic, histopathological, and molecular biological analyses were performed in the liver of male F344 rats fed 2 doses of FF (Experiment 1; 0 and 6000 ppm) for 3 weeks and 3 doses (Experiment 2; 0, 3000, and 6000 ppm) for 9 weeks. FF treatment increased the activity of enzymes such as carnitine acetyltransferase, carnitine palmitoyltransferase, fatty acyl-CoA oxidizing system, and catalase in the liver. However, it decreased those of superoxide dismutase in the liver in both experiments. Increased 8-hydroxy-2'-deoxyguanosine levels in liver DNA and lipofuscin accumulation were observed in the treated rats of Experiment 2. In vitro measurement of reactive oxygen species (ROS) in rat liver microsomes revealed a dose-dependent increase due to FF treatment. Microarray (only Experiment 1) or real-time reverse transcription-polymerase chain reaction analyses revealed that the expression levels of metabolism and DNA repair-related genes such as Aco, Cyp4a1, Cat, Yc2, Gpx2, Apex1, Xrcc5, Mgmt, Mlh1, Gadd45a, and Nbn were increased in FF-treated rats. These results provide evidence of a direct or indirect relationship between oxidative stress and FF treatment. In addition, increases in the expression levels of cell cycle-related genes such as Chek1, Cdc25a, and Ccdn1; increases in the expression levels of cell proliferation-related genes such as Hdgfrp3 and Vegfb; and fluctuations in the expression levels of apoptosis-related genes such as Casp11 and Trp53inp1 were observed in these rats. This suggests that cell proliferation induction, apoptosis suppression, and DNA damage due to oxidative stresses are probably involved in the mechanism of hepatocarcinogenesis due to FF in rats.

OBJECTIVE

Transforming growth factor beta (TGF-beta) is a multifunctional cytokine that strongly inhibits epithelial cell growth. Disabling of TGF-beta signaling is thought to be involved in development of a variety of tumors in which abnormal expression or function of TGF-beta receptor plays critical roles. In the present study, we examined aberrant expression and mutation of the gene TGF-beta receptor type II (TbetaRII) in endometrial cancers of endometrioid subtype.

RESULTS

Real-time PCR analysis using surgical tissue specimens of 27 endometrial cancers and 24 normal endometria revealed that endometrial cancers had significantly decreased levels of TbetaRII mRNA expression (mean level 2.44 +/- 2.65), compared to normal endometria (mean level 7.23 +/- 6.07) (P < 0.001). Methylation status of TbetaRII promoter containing 30 CpGs was examined by bisulfite sequencing analysis, and 98% (51/52) of the patients were found to have unmethylated TbetaRII promoter, indicating that promoter hypermethylation is not the major cause of decreased expression of TbetaRII in endometrial cancers. Mutational analysis revealed that 15.1% (8/53) of endometrial cancers had frameshift mutations at polyadenine repeats in exon 3 of the TbetaRII gene. Notably, these mutations were preferentially accumulated in patients with MSI-H phenotype (7/19:37%) (P < 0.001) or with those with methylated MLH1 promoters (6/16:38%) (P < 0.01). Thus, it appears that the TbetaRII gene is a target of mismatch repair deficiency.

CONCLUSIONS

Taken together, we found that the decreased expression of TbetaRII as well as frameshift mutation of TbetaRII via mismatch repair deficiency frequently occurs in this tumor type, possibly causing loss of receptor function and unresponsiveness of TGF-beta signaling that may lead to endometrial carcinogenesis.