MSI-positive Tumors Research Articles

Genetic Mechanisms in Interval Colon Cancers

The factors underlying the development of interval colon cancers are not well defined and are likely heterogeneous. We sought to determine whether there are distinct molecular properties associated with interval colon cancers. Colon cancers diagnosed within 5 years of a complete and well-prepped colonoscopic examination were identified over a 7-year period at a single institution. The clinical and pathological features of the tumors were defined. Analysis of DNA mismatch repair (MMR) and genotyping of a panel of oncogenes associated with colon cancer were performed. Forty-two interval colon cancers were diagnosed at an average age of 70 years. 69 % of tumors were located in the right colon. 41 % of tumors exhibited DNA microsatellite instability (MSI). Loss of staining of DNA MMR proteins by immunohistochemistry (IHC) was confirmed in 82 % of the MSI-positive tumors. Among tumors with abnormal MSI and IHC, 54 % exhibited somatic methylation of the MLH1 promoter, but the remaining 43 % exhibited molecular features indicative of underlying Lynch syndrome (LS). The frequency of somatic mutations in the KRAS, BRAF, NRAS, and PIK3CA oncogenes was similar between interval cancer cases and controls. Interval colon cancers are not distinguished by the activation of the KRAS, NRAS, BRAF, or PIK3CA oncogenic pathways. However, MSI pathway defects are present in a significant proportion of interval colon cancers. Underlying LS may explain nearly half of these MSI-positive cases, and the remaining cases appear to represent sporadic serrated pathway tumors.

Somatic Mutations in MLH1 and MSH2 Are a Frequent Cause of Mismatch-Repair Deficiency in Lynch Syndrome-Like Tumors

Lynch syndrome is caused by germline mutations in the mismatch repair (MMR) genes. Tumors are characterized by microsatellite instability (MSI). However, a considerable number of MSI-positive tumors have no known molecular mechanism of development. By using Sanger and ion semiconductor sequencing, 25 MSI-positive tumors were screened for somatic mutations and loss of heterozygosity in mutL homolog 1 (MLH1) and mutS homolog 2 (MSH2). In 13 of 25 tumors (8 MLH1-deficient and 5 MSH2-deficient tumors), we identified 2 somatic mutations in these genes. We conclude that 2 acquired events explain the MMR-deficiency in more than 50% of the MMR-deficient tumors without causal germline mutations or promoter methylation.

Ataxia‐telangiectasia‐mutated protein expression with microsatellite instability in gastric cancer as prognostic marker

The prognostic significance of ataxia-telangiectasia-mutated (ATM) expression in gastric cancer remains unclear. The functional loss of ATM gene exhibits a biologic correlation with microsatellite instability (MSI). In this study, we investigated the significance of ATM expression with MSI by evaluating gastric cancer patients who had underwent curative resection. ATM expression was classified into low ATM expression (-, ±, +) and high ATM expression (++, +++) using immunohistochemistry analysis. MSI status was classified as MSI-negative (MSS, MSI-low) and MSI-positive (MSI-high). Of 321 patients, 205 (63.9%) exhibited low ATM expression and 116 (36.1%) exhibited high ATM expression. Low ATM expression was more frequently identified in patients of older age, more advanced stage and with MSI-positive tumor (p = 0.025, p = 0.001 and p = 0.014, respectively). The probability of 5-year disease-free survival (DFS) and overall survival (OS) was lower in low ATM expression group compared with the high ATM expression group (DFS: 62.5%, 76.4%, p = 0.017, OS: 65.9%, 78.5%, p = 0.027, respectively). According to MSI status, a subgroup of MSI-negative and low ATM expression cases exhibited the worst prognosis for DFS and OS; this subgroup also exhibited poorer DFS according to multivariable analysis (hazard radio = 1.8, 95% confidence interval, 1.2-2.8, p = 0.010), although prognostic value of ATM expression alone did not remain in the multivariable analysis. Taken together, these findings indicate that ATM expression with MSI status is an independent factor for gastric cancer prognosis in gastric cancer patients who received curative surgery.

Microsatellite Instability in Sarcoma: Fact or Fiction?

Microsatellite instability (MSI) is a unique molecular abnormality, indicative of a deficient DNA mismatch repair (MMR) system. Described and characterized in the colorectal cancer literature, the MSI-positive phenotype is predictive of disease susceptibility, pathogenesis, and prognosis. The clinical relevance of MSI in colorectal cancer has inspired similar inquisition within the sarcoma literature, although unfortunately, with very heterogeneous results. Evolving detection techniques, ill-defined sarcoma-specific microsatellite loci and small study numbers have hampered succinct conclusions. The literature does suggest that MSI in sarcoma is observed at a frequency similar to that of sporadic colorectal cancers, although there is little evidence to suggest that MSI-positive tumors share distinct biological attributes. Emerging evidence in Ewing sarcoma has demonstrated an intriguing mechanistic role of microsatellite DNA in the activation of key EWS/FLI-target genes. These findings provide an alternative perspective to the biological implications of microsatellite instability in sarcoma and warrant further investigation using sophisticated detection techniques, sensitive microsatellite loci, and appropriately powered study designs.

Clinical Outcomes of Patients with Microsatellite-Unstable Colorectal Carcinomas Depend on L1 Methylation Level

The prognostic significance of microsatellite instability (MSI) in colorectal cancers (CRCs) has been addressed in many studies since the initial description of better survival rates in MSI-positive (MSI+) tumors than in MSI-negative (MSI-) tumors. Recent studies have demonstrated that a higher degree of hypomethylation of long interspersed nuclear element-1 (L1) is related to poor prognosis of CRCs and that a wide variation of L1 methylation levels exist within MSI+ CRCs. Our aim was to identify whether L1 and Alu methylation status could predict clinical outcomes within MSI+ CRCs. We analyzed 207 MSI+ CRCs for their methylation levels in L1 and Alu repetitive DNA elements using pyrosequencing and correlated them with clinicopathological information including survival data. Univariate survival analysis showed that low Alu methylation status (<18.60%) and low L1 methylation status (<53.00%) were significantly associated with shorter overall survival time (log-rank test, P = 0.009 and P < 0.001, respectively). Multivariate analysis using nine parameters (Alu methylation status, L1 methylation status, patient's age, disease stage [tumor, node, metastasis staging system], differentiation, Crohn-like lymphoid reaction, KRAS/BRAF mutation status, CpG island methylator phenotype [CIMP] status, and peritumoral lymphocytic infiltration), which were significantly prognostic in MSI+ CRCs, revealed that low L1 methylation status was an independent prognostic factor of MSI+ CRCs (P = 0.009), whereas low Alu methylation status was not. Clinical outcomes of MSI+ CRCs depend on L1 methylation status, suggesting that lower L1 methylation status serves as a significant prognostic parameter of adverse prognosis in MSI+ CRCs.

Abstract 3768: Colorectal cancer risk is not associated with increased levels of homozygosity in a population from Saudi Arabia

Abstract Autozygosity is a term used to denote the presence of two identical haplotypes that are derived from an ancestor shared by both parents, so it essentially represents a special type of homozygosity. Runs of homozygosity (ROH) in the genome is a measure of the extent of autozygosity and is directly correlated with the extent of inbreeding. While the role of ROH in unmasking recessively acting mutations is well established in Mendelian genetics, much less is known about their contribution to more complex disorders such as cancer. Recently, it has been suggested that ROH may contribute to the risk of colorectal cancer (CRC) perhaps through the unmasking of a recessively acting CRC-predisposing mutations in one or more genes. However, this observation could not be replicated. In this study, we examine the role of homozygosity in the CRC risk by asking four specific questions. First, do CRC patients have enrichment for ROH in particular chromosomal regions compared to controls? Second, do CRC patients have longer ROH compared to controls? Third, is there a particular SNP that is more likely to be homozygous in CRC patients compared to controls? And fourth, are CRC patients more inbred than controls? By comparing 48 Saudi CRC patients to 100 ethnically matched controls, all processed on Affy 250SytI SNP Chip platform and analyzed by Partek, we found that the answer is no to all these four questions. We note here that this is the first study to address these questions in an inbred population so the negative results in our study carry more significance than what has been previously reported in the literature. We also note that our subgroup analysis of patients with MSI-positive tumors compared to other groups did not significantly change our results. While these results do not rule out the potential presence of recessively acting CRC-predisposing genes in a small percentage of patients that our relatively small sample size could not capture, they do suggest that such genes are unlikely to account for the disturbingly high incidence of CRC in our consanguineous population and that future research should consider other mechanisms. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3768. doi:10.1158/1538-7445.AM2011-3768

Abstract 2152: Genome-wide single nucleotide polymorphism arrays in endometrial carcinomas associate extensive chromosomal instability with poor prognosis and unveil prevalent alterations in Ras/PI3-kinase pathway

Abstract [Objectives]: Endometrial cancer is one of the tumor types, in which either chromosomal instability (CIN) or microsatellite instability (MSI) may occur, and is known to possess mutations frequently in Ras-PI3K (phosphatidylinositol 3’-kinase) pathway. In this study, we attempted to clarify prognostic impact of genomic instability, determined by CIN and MSI status, and figure out the relationship between alterations in the Ras-PI3K pathway genes and the status of genomic instability. [Materials and Methods]: Under the approval of institutional ethical committees, we analyzed allele-specific chromosomal copy number alterations by SNP typing arrays in 31 endometrioid endometrial adenocarcinomas with paired DNA (25 by the 50K and six by the 250K probes). We also screened 25 of the 31 samples for MSI status with five microsatellite markers, and 22 samples for mutations in PIK3CA (exon 9 and 20), PTEN (exons 1-8) and K-Ras (exon1 and 2). Survival curves were constructed using the Kaplan-Meier method and compared with a log-rank test. [Results]: We detected five or more copy number changes (classified as CIN-extensive) in nine (29%), one to four changes (CIN-intermediate) in 17 (55%) and no changes (CIN-negative) in five (16%) tumors. Positive MSI was detected in ten (40%) of the 25 tumors and was less common in CIN-extensive tumors (14%), compared with CIN-intermediate/negative tumors (50%). CIN-extensive was significantly poor prognostic (p=0.0034), and the prognosis was shown to be independent from any other clinicpopathological characteristics by multivariate analysis. In contrast, positive MSI showed a trend to be a favorable prognostic factor (p=0.069), in spite of the association with vascular invasion (p=0.049). SNP array analysis unveiled copy number neutral LOH at 54 loci in 13 tumors (42%), including four at the locus of PTEN, and homozygous deletions at five regions in three tumors, including the locus of PTEN and NF1. Totally we detected eight (26%) tumors with PTEN deletions, and four (13%) with NF1 deletions. We identified mutations of PTEN, PIK3CA and K-Ras in 16 (73%), 6 (27%) and 2 (9%) out of 22 tumors, respectively. Seven (78%) of the nine CIN-extensive tumors harbor deletions at the loci of PTEN and/or NF1, with less frequent PTEN mutations (22%). On the other hand, PTEN mutations were detected in all the ten MSI-positive tumors, five of which coexist with PIK3CA and/or K-Ras mutations. We detected no deletions of NF1 and only one (10%) LOH of PTEN in the ten MSI-positive tumors. [Conclusion]: Our results demonstrated that the degree of CIN is a useful biomarker for prognosis in endometrial carcinomas. In addition, genomic alterations in the Ras-PI3K pathway are remarkably widespread in endometrial carcinomas, regardless of the type of genomic instability. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2152.

Genome-wide single-nucleotide polymorphism arrays in endometrial carcinomas associate extensive chromosomal instability with poor prognosis and unveil frequent chromosomal imbalances involved in the PI3-kinase pathway

Endometrial cancer is one of the tumor types in which either chromosomal instability (CIN) or microsatellite instability (MSI) may occur. It is known to possess mutations frequently in the Ras-PI3K (phosphatidylinositol 3'-kinase) pathway. We performed a comprehensive genomic survey in 31 endometrial carcinomas with paired DNA for chromosomal imbalances (25 by the 50K and 6 by the 250K single-nucleotide polymorphism (SNP) array), and screened 25 of the 31 samples for MSI status and mutational status in the Ras-PI3K pathway genes. We detected five or more copy number changes (classified as CIN-extensive) in 9 (29%), 1 to 4 changes (CIN-intermediate) in 17 (55%) and no changes (CIN-negative) in 5 (16%) tumors. Positive MSI was less common in CIN-extensive tumors (14%), compared with CIN-intermediate/negative tumors (50%), and multivariate analysis showed that CIN-extensive is an independent poor prognostic factor. SNP array analysis unveiled copy number neutral LOH at 54 loci in 13 tumors (42%), including four at the locus of PTEN. In addition to eight (26%) tumors with PTEN deletions, we detected chromosomal imbalances of NF1, K-Ras and PIK3CA in four (13%), four (13%) and six (19%) tumors, respectively. In all, 7 of the 9 CIN-extensive tumors harbor deletions in the loci of PTEN and/or NF1, whereas all the 10 MSI-positive tumors possess PTEN, PIK3CA and/or K-Ras mutations. Our results showed that genomic alterations in the Ras-PI3K pathway are remarkably widespread in endometrial carcinomas, regardless of the type of genomic instability, and suggest that the degree of CIN is a useful biomarker for prognosis in endometrial carcinomas.

ATR Mutation in Endometrioid Endometrial Cancer Is Associated With Poor Clinical Outcomes

PURPOSE Mutations in the DNA damage response gene ATR (exon 10 A10 mononucleotide repeat) have been previously described in endometrial and other cancers with defective DNA mismatch repair. In vitro studies showed that endometrial cancer cell lines with A10 repeat tract truncating mutations have a failure in the ATR-dependent DNA damage response. Cell lines carrying A10 mutations fail to trigger Chk1 activation in response to ionizing radiation and topoisomerase inhibitors. We sought to determine the frequency and clinicopathologic significance of ATR mutations in patients with endometrioid endometrial cancer. PATIENTS AND METHODS The ATR exon 10 A10 repeat was analyzed by direct sequencing in 141 tumors with microsatellite instability (MSI-positive) and 107 microsatellite stable (MSI-negative) tumors. The relationships between mutations and clinicopathologic variables, including overall and disease-free survival, were assessed using contingency table tests and Cox proportional hazard models. Results ATR mutations were identified in 12 cases (4.8%; three cases with insertions and nine cases with deletions). Mutations occurred exclusively in MSI-positive tumors (P = .02), with an overall mutation rate of 8.5%. Mutation was not associated with age, race, surgical stage, International Federation of Gynecology and Obstetrics grade, or adjuvant treatment. Multivariate analyses revealed a significant association with reduced overall survival (hazard ratio [HR] = 3.88; 95% CI, 1.64 to 9.18; P = .002) and disease-free survival (HR = 4.29; 95% CI, 1.48 to 12.45; P = .007). CONCLUSION Truncating ATR mutations in endometrial cancers are associated with biologic aggressiveness as evidenced by reduced disease-free and overall survival. Knowledge of ATR mutation status may hold promise for individualized treatment and targeted therapies in patients with endometrial cancer.

Effects of ovariectomy on microsatellite instability in rat colon tumors induced by 1,2-dimethylhydrazine

To study the effects of ovariectomy on tumorigenesis and microsatellite instability (MSI) in rat colon tumors induced by 1,2-dimethylhydrazine, to elucidate the association between postmenopausal ovarian hormones depletion and MSI pathway in colorectal tumorigenesis. Forty female Wistar rats were randomly divided into two groups: Ovariectomized (Ovx) group and Sham-ovariectomized (Sham-Ovx) group. All rats were injected intraperitoneally with 1,2-dimethylhydrazine (DMH) (20 mg/kg b.w) once a week for 20 weeks. Ten weeks after the final DMH injection, all the rats were sacrificed to collect tumors. Microsatellite instability of six microsatellite loci was detected using fluorescent PCR followed by fragment analysis on automatic DNA sequencer with GeneScan 3.7 software. The tumor multiplicity in the OVX group was significantly higher than that in the Sham-OVX group (3.6 +/- 1.4 vs. 2.4 +/- 1.6, P < 0.05). The incidence of MSI-positive tumors in OVX group was higher than that in Sham-OVX group (32.1 vs. 10.8%, P < 0.05).The incidence of tumors showing MSI at multiple loci in OVX group was also higher than that in Sham-OVX group (18.9 vs. 2.7%, P < 0.05). Ovariectomy increased tumor formation and the frequency of MSI in DMH-induced colon tumors. It implied that postmenopausal ovarian hormones depletion might influence colorectal tumorigenesis through MSI pathway.

Identification in Daily Practice of Patients With Lynch Syndrome (Hereditary Nonpolyposis Colorectal Cancer): Revised Bethesda Guidelines-Based ApproachVersusMolecular Screening

The identification of individuals who should undergo hereditary nonpolyposis colorectal cancer (HNPCC) genetic testing remains a critical issue. The Bethesda guidelines were developed to preselect patients for microsatellite instability (MSI) testing before germline mutation screening. These criteria have been revised, and a new set of recommendations, the revised Bethesda guidelines, has been proposed. To evaluate the performance of these revised guidelines for identifying patients with HNPCC in a series of unselected consecutive patients and compare this revised guidelines-based approach with a molecular strategy (MSI testing for all tumors, followed by exclusion of MSI-positive sporadic cases from mutational testing). The study included 214 patients with newly diagnosed colorectal cancer. The MSI analysis was performed for all tumors. Germline testing, guided by immunohistochemical staining for mismatch repair proteins, was performed for patients with MSI-positive tumors. Sporadic MSI-positive tumors were identified by screening for BRAF mutation and MLH1 promoter methylation. Ninety patients (42.1%) met the revised guidelines. Twenty-one patients (9.8%) had MSI-positive tumors. Germline testing identified eight mutations (3.7%) (MSH2 N = 5, MLH1 N = 2, MSH6 N =1). The revised guidelines failed to identify 2 of the 8 probands (aged 67 and 81 yr, both with no family history). In contrast, the molecular strategy identified all patients requiring testing for germline mutation. The percentages of patients selected for germline testing by the revised guidelines and the molecular strategy were 4.2% and 5.1%, respectively. The revised Bethesda guidelines did not identify all HNPCC cases in our series. The molecular approach identified all HNPCC patients with MSI-positive tumors, increasing the workload for germline testing only slightly.

Exclusive KRAS mutation in microsatellite-unstable human colorectal carcinomas with sequence alterations in the DNA mismatch repair gene, MLH1

Microsatellite instability (MSI) is regarded as reflecting defective DNA mismatch repair (MMR). MMR defects lead to an increase in point mutations, as well as repeat instability, on the genome. However, despite the highly unstable microsatellites, base substitutions in representative oncogenes or tumor suppressors are extremely infrequent in MSI-positive tumors. Recently, the heterogeneity in MSI-positive colorectal tumors is pointed out, and the ‘hereditary’ and ‘sporadic settings’ are proposed. Particularly in the former, base substitution mutations in KRAS are regarded as relatively frequent. We sequenced the KRAS gene in a panel of 76 human colorectal carcinomas in which the MSI status has been determined. KRAS mutations were detected in 22 tumors (28.9%). Intriguingly, all of the KRAS-mutant MSI-H (high) tumors harbored sequence alterations in an essential MMR gene, MLH1, which implies that KRAS mutation more frequently and almost exclusively occurs in MMR gene-mutant MSI-H tumors. Furthermore, in contrast with the prevailing viewpoint, some of these tumors are derived from sporadic colorectal cancer patients. The tight connection between MMR gene mutation and KRAS mutation may suggest previously unrecognized complexities in the relationship between MSI and the mutator phenotype derived from defective MMR.

Development of new immunochemical assays for DNA adducts, with high-throughput potential

The aim of this study was to assess the performance of immunohistochemistry using antibodies for MLH1, MSH2, MSH6 and PMS2 mismatch repair gene proteins against microsatellite instability (MSI) testing.Tumour samples included in this study were derived from referred patients for screening for hereditary non-polyposis colorectal cancer (HNPCC) and patients who had resections for colorectal cancer that were examined at our institution. MSI was assessed at nine loci (BAT25, BAT26, BAT40, D2S123, D10S197, D17S579, D18S34, D5S346 and D17S250) in all cases. Immunohistochemistry for MLH1 and MSH2 was performed in all cases. Staining for MSH6 and PMS2 was performed in selected cases only.There were 742 tumours including 661 colorectal lesions and 81 extracolonic tumours of the HNPCC spectrum. Among the 555 MSI-negative tumours, 554 showed an intact protein expression. Amongst the 187 MSI-positive tumours, 126 showed abnormal expression of MLH1 gene protein, 41 showed abnormal expression of MSH2 gene, three showed abnormal expression of MSH6 only, one showed abnormal expression of PMS2 gene protein only and one case showed abnormal expression of all four proteins.Immunohistochemistry offers an alternative method for assessment of MSI status which is fast and relatively inexpensive compared with MSI testing. We achieved a sensitivity rate of 92% and specificity of 99.8% for immunohistochemistry testing assessed against the MSI testing. It has to be accepted that a small fraction of MSI-positive cases will be missed by testing with immuno-histochemistry alone.

Lynch Syndrome (Hereditary Nonpolyposis Colorectal Cancer) Diagnostics

Preventive programs for individuals who have high lifetime risks of colorectal cancer may reduce disease morbidity and mortality. Thus, it is important to identify the factors that are associated with hereditary colorectal cancer and to monitor the effects of tailored surveillance. In particular, patients with Lynch syndrome, hereditary nonpolyposis colorectal cancer (HNPCC), have an increased risk to develop colorectal cancer at an early age. The syndrome is explained by germline mutations in DNA mismatch repair (MMR) genes, and there is a need for diagnostic tools to preselect patients for genetic testing to diagnose those with HNPCC. Patients (n = 112) from 285 families who were counseled between 1990 and 2005 at a clinic for patients at high risk for HNPCC were selected for screening to detect mutations in MMR genes MLH1, MSH2, MSH6, and PMS2 based on family history, microsatellite instability (MSI), and immunohistochemical analysis of MMR protein expression. Tumors were also screened for BRAF V600E mutations; patients with the mutation were considered as non-HNPCC. Among the 112 patients who were selected for screening, 69 had germline MMR mutations (58 pathogenic and 11 of unknown biologic relevance). Sixteen of the 69 mutations (23%) were missense mutations. Among patients with MSI-positive tumors, pathogenic MMR mutations were found in 38 of 43 (88%) of patients in families who met Amsterdam criteria and in 13 of 22 (59%) of patients in families who did not. Among patients with MSI-negative tumors, pathogenic MMR mutations were found in 5 of 17 (29%) of families meeting Amsterdam criteria and in 1 of 30 (3%) of non-Amsterdam families with one patient younger than age 50 years. In three patients with MSI-negative tumors who had pathogenic mutations in MLH1 or MSH6, immunohistochemistry showed loss of the mutated protein. Our findings suggest that missense MMR gene mutations are common in HNPCC and that germline MMR mutations are also found in patients with MSI-negative tumors.

Cyclin D1 G870A Variant is Associated with Increased Risk of Microsatellite Instability-Positive Colorectal Cancer in Young Male Patients

Cyclin D1 G870A Variant is Associated with Increased Risk of Microsatellite Instability-Positive Colorectal Cancer in Young Male PatientsCyclin D1 (CCND1) is a cell cycle regulatory protein, which is often over expressed in human tumors and is associated with cell proliferation and poor prognosis. A common G870A single nucleotide polymorphism at codon 242 in exon 4 of the CCND1 gene is associated with an altered messenger RNA transcript and increased risk of colorectal cancer (CRC) and adenoma in some studies. Over expression of CCND1 modifies the effect of mutations in mismatch repair (MMR) genes, enhances microsatellite instability (MSI), and influences the age ofonset of hereditary non polyposis colorectal cancer (HNPCC). We have extended our study that indicated that the CCND1 A variant may influence the age of onset of CRC in the Macedonian population only in patients who exhibit MSI tumors by a case control study of 331 randomly selected CRC patients and 101 controls without clinical diagnosis of CRC. We did not observe a significant difference in overall allelic frequencies and genotype distribution of affected and unaffected mutation carriers, but found a statistically significant risk of CRC in carriers of the CCND1 A allele when patients were grouped according to gender, age and MSI status. A higher risk was observed in patients with MSI-positive tumors and particularly in male patients under 60 years of age. The consequences of the above observation were reversed in female patients. These results indicate that the CCND1 A variant may enhance CRC progression through a pathway influenced by estrogens in colonic epithelia.

Screening for Lynch Syndrome (Hereditary Nonpolyposis Colorectal Cancer) among Endometrial Cancer Patients

Endometrial cancer is the most common cancer in women with Lynch syndrome. The identification of individuals with Lynch syndrome is desirable because they can benefit from increased cancer surveillance. The purpose of this study was to determine the feasibility and desirability of molecular screening for Lynch syndrome in all endometrial cancer patients. Unselected endometrial cancer patients (N = 543) were studied. All tumors underwent microsatellite instability (MSI) testing. Patients with MSI-positive tumors underwent testing for germ line mutations in MLH1, MSH2, MSH6, and PMS2. Of 543 tumors studied, 118 (21.7%) were MSI positive (98 of 118 MSI high and 20 of 118 MSI low). All 118 patients with MSI-positive tumors had mutation testing, and nine of them had deleterious germ line mutations (one MLH1, three MSH2, and five MSH6). In addition, one case with an MSI-negative tumor had abnormal MSH6 immunohistochemical staining and was subsequently found to have a mutation in MSH6. Immunohistochemical staining was consistent with the mutation result in all seven truncating mutation-positive cases but was not consistent in two of the three missense mutation cases. We conclude that in central Ohio, at least 1.8% (95% confidence interval, 0.9-3.5%) of newly diagnosed endometrial cancer patients had Lynch syndrome. Seven of the 10 Lynch syndrome patients did not meet any published criteria for hereditary nonpolyposis colorectal cancer, and six of them were diagnosed at age >50. Studying all endometrial cancer patients for Lynch syndrome using a combination of MSI and immunohistochemistry for molecular prescreening followed by gene sequencing and deletion analysis is feasible and may be desirable.

Author reply

The letter by Vilar et al. gives us the opportunity to highlight the consequences our recent characterization of the BRAF V599E (V600E) mutation in colorectal cancer patients has had on the diagnosis of hereditary nonpolyposis colorectcal carcinoma (HNPCC). The first step is still analysis of the family history.1 Patients meeting the Amsterdam criteria are recommended for sequencing of the mismatch repair (MMR) genes MLH1 and MSH2.2 If the Amsterdam criteria are not fulfilled or familial data are unavailable, the Bethesda guidelines help to select patients for testing microsatellite instability (MSI), a hallmark of HNPCC that also occurs in some sporadic colorectal cancers.3 If MSI analysis is negative, HNPCC can be excluded. In colorectal cancer patients with MSI, the immunohistochemistry for MLH1 and MSH2 provides information regarding which MMR gene may be mutated. However, negative immunohistochemistry for MLH1 also may be caused by epigenetic gene silencing. Our recent article demonstrated that the latter is frequently accompanied by the BRAF V599E mutation.4 Therefore, at this stage of HNPCC diagnosis, the presence of the BRAF V599E mutation allows us to exclude patients whose loss of MLH1 has an epigenetic background. Because our study data demonstrated that the BRAF V599E mutation also is associated with an older age at the time of diagnosis, we would especially recommend this diagnostic step in older patients. Screening for the BRAF V599E mutation also may be performed in all MSI-positive tumors instead of immunohistochemistry, but to our knowledge, studies evaluating this diagnostic procedure are not yet available. Nikolaus Lubomierski MD*, Guido Plotz PhD*, Jochen Raedle MD*, * Department of Internal Medicine II, University of the Saarland, Hamburg, Germany.

Microsatellite Instability Caused by hMLH1 Promoter Methylation Increases with Tumor Progression in Right-Sided Sporadic Colorectal Cancer

Objective: A subset of sporadic colorectal cancers (SCRCs) exhibits microsatellite instability (MSI). Most MSI in SCRCs is caused by hMLH1 inactivation due to promoter methylation. However, the role of MSI in the progression of SCRCs remains unclear. Methods: Thirty-two intramucosal cancers and 63 cancers with submucosal invasion were assigned to group 1 (early-stage cancer), and 30 Dukes’ B and 26 Dukes’ C cancers to group 2 (advanced-stage cancer). hMLH1 promoter methylation status was determined by methylation-specific PCR. MSI was determined using five markers. hMLH1 expression was determined immunohistochemically. Results: MSI was found in 1 of 95 (1.1%) tumors in group 1, compared with 4 of 56 (7.1%) tumors in group 2. In right-sided tumors, the overall frequency of hMLH1-methylation-positive tumors in group 1 was not significantly different from that in group 2 (17 of 43, 39.5%, vs. 9 of 23, 39.1%). In right-sided tumors with hMLH1 promoter methylation, the frequency of MSI-positive tumors in group 1 was significantly lower than that in group 2 (1 of 17, 5.9%, vs. 4 of 9, 44.4%, p = 0.0081). Conclusion: The frequency of MSI caused by hMLH1 promoter methylation increases with tumor progression in right-sided SCRCs.

Molecular Genetic Alterations and Clinical Features in Early-Onset Colorectal Carcinomas and Their Role for the Recognition of Hereditary Cancer Syndromes

Colorectal cancer (CRC) occurs rarely in young individuals (<45 yr) and represents one of the criteria for suspecting hereditary cancer families. In this study we evaluated clinical features and molecular pathways (chromosomal instability [CIN] and microsatellite instability [MSI]) in early-onset CRC of 71 patients. Detailed family and personal history were obtained for each patient. Expression of APC, beta-catenin, p53, MLH1, MSH2, and MSH6 genes was evaluated by immunohistochemistry. MSI analysis was performed and constitutional main mutations of the mismatch repair (MMR) genes were searched by gene sequencing. Fourteen (19.7%) out of the 71 cases showed both MSI and altered expression of MMR proteins. In the 57 MSI-negative (MSI-) lesions altered expression of APC, beta-catenin, and p53 genes were found more frequently than in MSI-positive(MSI+) tumors. Seven (50%) out of the 14 patients with MSI+ tumors presented clinical features of Lynch syndrome (hereditary non-polyposis colorectal cancer [HNPCC]) and in all but one, constitutional mutations in MLH1 or MSH2 genes could be detected. The same mutations were also found in other family members. Our study demonstrates the involvement of CIN in a majority of early-onset colorectal tumors. Furthermore, we identified Lynch syndromes in seven cases (50%) of early-onset colorectal carcinomas with impairment of the MMR system. These results suggest that patients with early-onset CRC should be screened for hereditary cancer syndrome through clinical and molecular characterizations.

MSI in endometrial cancer: Prevalence and clinical applications

Background: Loss of DNA mismatch repair is a frequent event in endometrial cancers, leading to an easily recognizable molecular phenotype referred to as microsatellite instability (MSI). In colorectal cancers MSI appears to be of prognostic significance. The relationship between MSI and outcome and other clinicopathologic variables in endometrial cancer has not been firmly established. Objectives: Assess the relationship between tumor MSI and clinicopathologic variables in our large cohort of endometrial cancer cases, and compare our findings with previous reports on MSI in endometrial cancers. Methods: 600 hundred prospectively acquired uterine cancers were assessed for MSI using 5 microsatellite repeat markers and MSI status classified using standard conventions. All MSI-positive tumors were evaluated for methylation of the MLH1 DNA mismatch repair gene promoter. Information on patient age at diagnosis, tumor histology and stage were obtained at time of surgery. For 411 patients treated in the period 1991–2001, medical records were reviewed for evidence of other malignancies. Follow-up for 178 patients with endometrioid adenocarcinomas treated in the period 1992–1999 was completed mid-2004 by contacting patients and/or their physicians. Detailed family histories of cancer were developed for 164 probands in the series. Results: MSI was seen in 28% of the endometrial tumors in our series. There was a significant association between MSI and endometrioid histology but not with any of the other clinicopathologic variables investigated. However, when MSI was further classified according to MLH1 promoter methylation status, clear associations between high-level MSI in the absence of MLH1 methylation and clinical variables became evident. Women with MSI-positive tumors lacking MLH1 methylation were younger than women with tumors that showed MLH1 methylation. Furthermore, women with MSI-positive tumors lacking MLH1 methylation had significantly increased risk for second tumors that are associated with the hereditary nonpolyposis colorectal cancer syndrome. Women with MSI-positive tumors that lack methylation frequently harbor inherited mutations in DNA mismatch repair. There was no association between tumor MSI and survival for patients with endometrioid endometrial cancers. Conclusions: MSI is a common defect in endometrial cancers. MSI in the absence of MHL1 methylation may point to women who are at increased risk for second cancers associated with the hereditary nonpolyposis colorectal cancer syndrome. Typing tumors for MSI and MLH1 methylation coupled with immunohistochemistry to assess mismatch repair gene expression may prove useful in identifying women with inherited forms of endometrial cancer.