Low-level Microsatellite Instability Research Articles

Tu1682 Defective DNA Mismatch Repair Activity Is Common in Sporadic Sebaceous Neoplasms, and an Ineffective Approach to Screen for Lynch Syndrome

(Background) We have demonstrated that down-regulation of hMSH3 results in low levels of microsatellite instability (MSI-L) and elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) in tissue cultured cell lines. Primary tumor tissues exhibiting MSIL and/or EMAST from stage II/III CRC show intra-tumor depression of hMSH3 and upregulation of GLUT1 that is a marker for hypoxia. In vitro, hypoxia induces down-regulation of hMSH3 and the development of EMAST in CRC cell lines. Finally, Stage II/III primary CRC exhibitingMSI-L/EMAST has a shorter recurrence-free survival than that of microsatellite stable counterparts not exhibiting MSI-L/EMAST. These results support the concept that intra-tumor hypoxia may induce MSI-L/EMAST through down-regulation of hMSH3 and enhance the aggressiveness of primary CRC, leading to recurrence and/or distant metastasis. Thus, MSI-L/EMAST could be a marker for identifying genetic and/or epigenetic changes that control recurrence and/or distant metastasis from primary CRC. We previously reported that LOH at the SMARCA2 locus on 9p24.3 is significantly high in liver metastasis (LM) exhibiting MSI-L/EMAST (termed moderate MSI: MSI-M) from primary CRC. (Aim) The purpose of this study is to determine the pathobiological significance of MSI-L/EMAST and LOH at SMARCA2 region in CRC. (Methods) Eighty three LMs and 186 primary CRC with known clinicopathological factors and disease outcome were analyzed for MSI/EMAST and LOH at the 9p24.2 region. Significant associations between factors were determined by various statistical analyses. (Results) We proposed that a locus affected by 9p24.2 LOH might promote metastasis from primary CRC; such a locus was mapped to an 80 kb region at the 3' boundary of the SMARCA2 gene. Stage II and III primary CRCs exhibiting MSIM and 9p24.2 LOH were significantly more likely to give rise to a solitary LM suitable for hepatectomy, whereas MSI-M-positive counterparts without 9p24.2 LOH, or highly microsatellite stable (HMSS) tissues were more likely to give rise to simultaneous metastases to multiple organs, and therefore, not suitable for surgical management. Multivariate analysis showed that MSI-M with 9p24.2 LOH in the primary CRC is an independent positive prognostic factor following recurrence (HR = .1981, P=.0007). (Conclusions) The presence or absence of 9p24.2 LOH in Stage II/III primary CRCwithMSI-M is an important determinant of the aggressiveness of the recurrent metastasis.

Analysis of microsatellite instability and loss of heterozygosity in ovarian cancer: a study in the population of Espírito Santo, Brazil

Ovarian cancer is currently the most lethal gynecological malignancy in women. It is a heterogeneous and cytogenetically complex disease previously associated with genomic instability. Our purpose was to analyze microsatellite markers to determine patterns and levels of instability as well as possible correlations with histopathological parameters. Polymerase chain reaction was used to characterize microsatellite instability (MSI) and loss of heterozygosity (LOH) in 24 ovarian tumors at 12 microsatellite loci. A total of 11 samples displayed MSI or LOH. Only low-level MSI was found. Markers D5S346 and CYP11 showed the highest MSI and LOH frequencies. D17S250 LOH was significantly associated with tumor histological type (P = 0.0003), and estrogen receptor α was also associated with tumor histological type (P = 0.048) when a combined analysis of LOH and MSI was performed. Furthermore, LOH was observed in a greater number of markers compared with those displaying MSI. Thus, our results support that MSI is less common than LOH in ovarian cancers.

Association of microsatellite instability with prognosis in the pathologic N2 colon cancer patients treated with adjuvant FOLFOX chemotherapy.

3568 Background: The impact of microsatellite instability (MSI) on survival has been rarely explored, with controversy, in stage III colon cancer treated with adjuvant 5-flurouracil-oxaliplatin combination (FOLFOX) chemotherapy. We evaluated association between MSI and survival in this population. Methods: We analyzed 312 patients (pts) with curatively resected stage III colon cancer treated with adjuvant FOLFOX chemotherapy. We determined MSI status using polymerase chain reaction amplification as high levels of MSI (MSI-H, ≥ 2 unstable markers), low levels of MSI (MSI-L, 1 unstable marker), and microsatellite stable (MSS, no unstable marker). Results: Of 312 pts, 8.3% showed MSI-H and they were younger (median age, 51.5 vs. 58 years, p=0.018). MSI-H tumors were more commonly located ascending colon (46.2% vs. 23.1%, p=0.002) and had poorly differentiated features (30.8% vs. 5.2%, p<0.0001). After the median follow-up of 30.9 months, 3-year relapse-free (RFS) and overall survival (OS) rates were 76.1% and 91.7%, respectively. In univariate analysis, pathologic N2 (pN2) was associated with reduced RFS (p<0.0001) and OS (p=0.002), while MSI status did not affect either RFS (p=0.254) or OS (p=0.961). In patients with pN2 tumors, however, MSI-H was associated with better survival compared with MSS/MSI-L; the RFS and OS in pts with pN2/MSI-H were comparable to those in pts with pN1 tumors (table). Conclusions: MSI status alone did not affect survival in our population. However, pts with pN2/MSI-H showed favorable survival outcomes comparable to those with pN1, and MSS/MSI-L/pN2 was associated with worst survival, implicating that MSI-H modifies the inherent worse prognosis of pN2 tumors. [Table: see text]

Association Between Recurrent Metastasis From Stage II and III Primary Colorectal Tumors and Moderate Microsatellite Instability

Colorectal cancer cells frequently have low levels of microsatellite instability (MSI-L) and elevated microsatellite alterations at selected tetranucleotide repeats (EMAST), but little is known about the clinicopathologic significance of these features. We observed that patients with stage II or III colorectal cancer with MSI-L and/or EMAST had shorter times of recurrence-free survival than patients with high levels of MSI (P = .0084) or with highly stable microsatellites P = .0415), based on Kaplan-Meier analysis. MSI-L and/or EMAST were independent predictors of recurrent distant metastasis from primary stage II or III colorectal tumors (Cox proportional hazard analysis: hazard ratio, 1.83; 95% confidence interval, 1.06-3.15; P = .0301).

Down-regulation of MutS homolog 3 by hypoxia in human colorectal cancer

Down-regulation of hMSH3 is associated with elevated microsatellite alterations at selected tetranucleotide repeats and low levels of microsatellite instability in colorectal cancer (CRC). However, the mechanism that down-regulates hMSH3 in CRC is not known. In this study, a significant association between over-expression of glucose transporter 1, a marker for hypoxia, and down-regulation of hMSH3 in CRC tissues was observed. Therefore, we examined the effect of hypoxia on the expression of hMSH3 in human cell lines. When cells with wild type p53 (wt-p53) were exposed to hypoxia, rapid down-regulation of both hMSH2 and hMSH3 occurred. In contrast, when null or mutated p53 (null/mut-p53) cells were exposed to hypoxia, only hMSH3 was down-regulated, and at slower rate than wt-p53 cells. Using a reporter assay, we found that disruption of the two putative hypoxia response elements (HREs) located within the promoter region of the hMSH3 abrogated the suppressive effect of hypoxia on reporter activity regardless of p53 status. In an EMSA, two different forms of HIF-1α complexes that specifically bind to these HREs were detected. A larger complex containing HIF-1α predominantly bound to the HREs in hypoxic null/mut-p53 cells whereas a smaller complex predominated in wt-p53 cells. Finally, HIF-1α knockdown by siRNA significantly inhibited down-regulation of hMSH3 by hypoxia in both wt-p53 and mut-p53 cells. Taken together, our results suggest that the binding of HIF-1α complexes to HRE sites is necessary for down-regulation of hMSH3 in both wt-p53 and mut-p53 cells.

Aberrant protein expression and frequent allelic loss of MSH3 in colorectal cancer with low-level microsatellite instability

High level of microsatellite instability (MSI-H) in colorectal cancer (CRC) is caused by the inactivation of mismatch repair (MMR) genes; however, it is unknown for tumors with low level MSI (MSI-L). The protein complex involving MSH3 preferentially recognizes insertion/deletion loops (IDLs) of two to eight bases and di- and tetranucleotide repeats are affected in the majority of MSI-L CRC. We selected 10 and eight MSI-L CRCs from 228 and 204 patients with sporadic and hereditary disease, respectively. The tumors were analyzed for protein expression of MSH3, MSH2, MSH6, MLH1, and PMS2, and for mutations and loss of heterozygosity (LOH) in MSH3. Four tumors showed a markedly reduced MSH3 expression, whereas all 18 tumors had normal expression of the remaining MMR proteins. Twenty-five different sequence variants were identified. None of these results in a truncated protein, though L902W represents the first constitutional missense mutation in MSH3 predicted to be functional based on conservation among mutS homologues. All variants have also been found in normal DNA of the patients and in controls. LOH intragenic to MSH3 was evident for 12 of 16 (75%) informative tumors. Occurrence of sequence variants in normal DNA of the patients and in controls excludes somatic mutations and mutations specific to the CRC patient population, respectively. In contrast, the high frequency of LOH as well as the aberrant protein expression in some tumors indicates an involvement of MSH3 impairment in MSI-L CRC.

Colorectal Cancer: Sailing with a T-Cell EMAST

During the past two decades, considerable progress has been made in understanding and defining the molecular basis of human colorectal cancer (CRC). The genetic heterogeneity of this disease is now well-established, and it has become clear that a distinct subgroup of CRC possesses a unique form of genetic instability, defined as microsatellite instability (MSI). The MSI phenotype is a hallmark of defective DNA mismatch repair (MMR), and inactivation of MMR genes permits increased instability and accumulation of genomic alterations in the sequences containing mono[e.g.(A)n] and di-nucleotide [e.g. (CA)n] microsatellite repeats. Approximately 12–15% of CRC exhibit high frequency MSI (MSI-H), which includes the majority of Lynch syndrome cancers with germline mutation of a MMR gene and about 12% of sporadic tumors due to methylation-induced transcriptional silencing of the MLH1 gene [1]. The remaining 85% of CRCs either possess very low levels of MSI (MSI-L) or are microsatellite stable (MSS); however, the molecular basis of these tumors remains poorly understood. Almost a decade ago, Sidransky and colleagues described a distinct form of genetic instability present in respiratory tumors, which was termed as elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) [2]. In contrast to instability at the monoand di-nucleotide repeats seen in MSI CRCs, EMAST tumors are identified when the frameshift mutations occur at certain tetranucleotide repeat sequences [e.g. (AAAG)n or (ATAG)n]. Since its initial discovery in non-small cell lung cancers [2, 3], EMAST has subsequently been observed in several other human cancers, including skin [4], ovarian [5], endometrial [6], bladder [4, 7], prostate [8, 9] and colorectal [10–13]. Most microsatellite sequences (either mono-, dior tetra-nucleotide repeats) are present in the non-coding regions of DNA; however, small minorities of these tandem repeats exist within the coding sequences of growth regulatory genes that play a critical role in driving CRC pathogenesis. Frameshift mutations within this latter class of genes, such as observed with EMAST, have direct functional consequences for the growth and proliferation of tumor cells. The underlying molecular mechanism(s) causing EMAST remain unclear. The earliest reports in this regard proposed an association of EMAST with mutations in the p53 gene [3, 4]. It was suggested that exposure to environmental carcinogens may exacerbate this phenotype [14]. With the limited evidence at hand, EMAST appears to be distinct from the classic MSI signature that is frequently observed in Lynch syndrome and sporadic CRC. Whether EMAST contributes to tumor progression by specific geneinactivating mutations within repetitive coding sequences, as is the case with MSI-positive CRC, is a matter of ongoing investigation. Recently, it was shown that EMAST is present in the majority of sporadic CRCs [10]. Haugen et al. demonstrated that EMAST is associated with MSI-L and a deficiency in MSH3 protein expression in colorectal cell lines and tumor tissues [10]. Building upon this evidence, later on it was demonstrated that EMAST can be acquired during the adenoma to carcinoma transformation, as well as upon transformation of well-differentiated carcinomas to moderately and poorly differentiated CRCs [12]. Interestingly in this study, EMAST best correlated in CRCs with ulcerated features, and the authors suggested that inflammation may help drive EMAST [12]. A. Goel (&) Gastrointestinal Cancer Research Laboratory, Sammons Cancer Center and Baylor Research Institute, Baylor University Medical Center, 3500 Gaston Avenue, Suite H-250, Dallas, TX 75246, USA e-mail: ajay.goel@baylorhealth.edu

Association of low‐risk MSH3 and MSH2 variant alleles with Lynch syndrome: Probability of synergistic effects

Mutations in the MLH1 and MSH2 genes account for a majority of cases of families with Lynch Syndrome. Germ-line mutations in MSH6, PMS2 and MLH3 are responsible for disease in a minority of cases, usually associated with milder and variable phenotypes. No germ-line mutations in MSH3 have so far been associated with Lynch Syndrome, although it is known that impaired MSH3 activity leads to a partial defect in mismatch repair (MMR), with low levels of microsatellite instability at the loci with dinucleotide repeats in colorectal cancer (CRC), thus suggesting a role for MSH3 in carcinogenesis. To determine a possible role of MSH3 as predisposing to CRC in Lynch syndrome, we screened MSH3 for germ-line mutations in 79 unrelated Lynch patients who were negative for pathogenetic mutations in MLH1, MSH2 and MSH6. We found 13 mutant alleles, including silent, missense and intronic variants. These variants were identified through denaturing high performance liquid chromatography and subsequent DNA sequencing. In one Lynch family, the index case with early-onset colon cancer was a carrier of a polymorphism in the MSH2 gene and two variants in the MSH3 gene. These variants were associated with the disease in the family, thus suggesting the involvement of MSH3 in colon tumour progression. We hypothesise a model in which variants of the MSH3 gene behave as low-risk alleles that contribute to the risk of colon cancer in Lynch families, mostly with other low-risk alleles of MMR genes.

Abstract 2261: Prognostic significance of the low level microsatellite instability phenotype differs with race in colorectal adenocarcinoma

Abstract Purpose: Several studies have examined the prognostic value of microsatellite instability (low, MSI-L, and high, MSI-H) phenotypes in colorectal adenocarcinomas (CRCs), but none have specifically considered patient race/ethnicity to assess the value of MSI-L. Therefore, this study assessed the prognostic value of the MSI-L phenotype in African-American and non-Hispanic Caucasian (NHCA) CRC patients separately. Experimental Design: CRCs from 276 African-Americans and 377 NHCAs, collected at the hospitals of the University of Alabama at Birmingham and Morehouse School of Medicine, were assessed for microsatellite instability and p53 mutational status (exons 4 through 9). MSI was assessed at 4 CA-dinucleotide MS loci (Mfd 27, Mfd 41, Mfd 47, and Mfd 57) and 2 poly-A repeats (BAT25, BAT26). Samples were considered MSI-H if at least 2 loci showed instability and MSI-L (low) if only 1 marker was unstable. The phenotypes were correlated with clinicopathologic features and patient survival by use of the χ2 test and univariate Kaplan-Meier analyses, respectively. Results: The incidence of the MSI-L phenotype was higher in African-American patients (49 of 276, 18%) compared to NHCA patients (27 of 377, 7%). In contrast, the frequency of the microsatellite stable (MSS) phenotype was higher in CRCs of NHCAs (72%) than in CRCs of African-Americans (56%). In addition, the incidence of microsatellite instability-high (MSI-H) phenotype was similar in African-Americans (26%) and NHCAs (21%). Presence of the MSI-L phenotype significantly correlated with a higher incidence of missense p53 mutations (64%) and high-grade tumors (31%) and with advanced stages (III and IV) (67%) of CRCs in NHCAs. Such associations, however, were not found in African-American patients. For NHCA patients, CRCs with the MSI-L phenotype had significantly higher mortality (log-rank p=0.044) as compared to MSS or MSI-H. In contrast, for African-American patients, the MSI-L phenotype was associated with a better prognosis (log-rank p=0.043) compared to the MSS or MSI-H phenotypes. Conclusions: These findings suggest that CRC with the MSI-L phenotype is associated with p53 mutations, high-grade tumors, and advanced tumor stage, and with short survival of NHCAs but not African-Americans. This work was supported by grants from the NCI/NIH (2U54-CA118948-03, R01-CA98932, and R03-CA139629). 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 2261. doi:10.1158/1538-7445.AM2011-2261

Genomic instability measured by inter-(simple sequence repeat) PCR and high-resolution microsatellite instability are prognostic of colorectal carcinoma survival after surgical resection.

During the multiyear progression to colorectal cancer, numerous genomic alterations arise in events ranging from single base mutations to gains or losses of entire chromosomes. A single genetic change might not stand out as an independent predictor of outcome. The goal of this study was to determine if more comprehensive measurements of genomic instability provide clinically relevant prognostic information. Our study included 65 sporadic colorectal cancer patients diagnosed from 1987 to 1991 with last follow-up ascertained in 2006. We estimated an overall tally of alterations using the genome-wide sampling technique of inter-(simple sequence repeat [SSR]) polymerase chain reaction (PCR), and evaluated its relationship with all-cause survival. We also extended and sensitized the Bethesda criteria for microsatellite instability (MSI), by analyzing 348 microsatellite markers instead of the normal five. We expanded the MSI categories into four levels: MSI stable (MSS), very low-level MSI, moderately low-level MSI, and classical high-level MSI. Tumors with genomic instability above the median value of 2.6% as measured by inter-SSR PCR, were associated with far greater risk of death compared to tumors with lower levels of genomic instability. Adverse outcome was most pronounced for patients presenting with stage 3 disease. A gradient of increased survival was observed across increasing MSI levels but did not reach statistical significance. Our findings suggest genomic instabilities quantified by inter-SSR PCR and increased precision in MSI values may be clinically useful tools for estimating prognosis in colorectal cancer.

Sporadic colorectal carcinomas with low-level microsatellite instability: a distinct subgroup with specific clinicopathological and molecular features

The biologic significance of low-level microsatellite instability (MSI) in sporadic colorectal cancers (CRCs) is not clearly defined. In particular, the relationship of MSI-low to MSI-high and microsatellite stable (MSS) tumours is currently under debate and the prognostic impact of these genetic changes remains unclear. The objective of this study was to investigate whether sporadic MSI-low CRCs have different clinicopathological and molecular features from MSS and MSI-high tumours. A series of 184 primary sporadic CRCs were divided, according to the level of MSI, into three groups (94 MSS, 22 MSI-low and 68 MSI-high) and were analyzed for baseline clinicopathological features and outcome, allelic losses at 18q, 8p and 4p chromosomes and immunohistochemical expression of MGMT, hMlh1, hMsh2, Fhit, Cox-2, p21 and p27 proteins. MSI-low tumours were more frequently distal (59.1%) whereas MSS tumours had a strong predilection for distal (72.3%) and MSI-high tumours for proximal location (54.4%; p = 0.003). When compared with MSI-high tumors, MSI-low CRCs were adenocarcinoma, not otherwise specified (p = 0.0138) and well to/moderately differentiated (p = 0.027). MSI-low CRCs also showed specific molecular features including intermediate 18q allelic losses, altered MGMT and Cox-2 expression. Finally, the 5-year overall survival rates were 79% for MSI-low, 40.3% for MSS and 71% for MSI-high CRCs (p = 0.0160 MSS vs. MSI-low groups). Sporadic MSI-low CRCs display characteristic clinicopathological and genetic features that distinguish them from MSS CRCs.

A microsatellita-státus és a morfológiai kép összefüggése vastagbélrákokban

Microsatellite instability (MSI) influences the development and clinical course of colorectal cancers (CRCs) and induce specific morphological alterations of such neoplasms, therefore hematoxylin-eosin (H&E) based histology allows to predict the microsatellite status of a given tumor. The aim of this article is to demonstrate clinicopathological features that are useful in recognition of microsatellite-stable and -unstable CRCs on routine histological examination. In the Center of Surgical and Molecular Pathology of National Institute of Oncology, from 384 CRC cases 26 hereditary non-polyposis colorectal cancers (HNPCC), 22 sporadic high-level microsatellite-instable (MSI-H) cancers and 76 microsatellite-stable (MSS) or low-level MSI (MSI-L) CRCs were selected on the basis of the localization, clinical stage, microsatellite status, and patient age at the time of the diagnosis. Our results showed that we can recognize MSS/MSI-L carcinomas, HNPCCs and sporadic MSI-H tumors with high probability on the base of clinicopathological features like patient's age, tumor localization, clinical stage and histological characteristics of CRCs, even if the genetic MSI test is not available. The main morphological characteristics related to microsatellite instability are intratumoral or stromal infiltrating lymphocytes/leukocytes, large, vesicular nuclei with prominent nucleoli, and expansive infiltrative edge of the tumors. Careful and detailed morphological analysis of colorectal cancers helps to select the appropriate molecular method to determine the molecular features that influence the clinical care of patients and allow to consider the most appropriate anti-tumor therapy.

Report From the Jerusalem Workshop on Lynch Syndrome-Hereditary Nonpolyposis Colorectal Cancer

A Workshop was held in Jerusalem, Israel, on October 26 and 27, 2009 to discuss the management of Lynch syndrome-hereditary nonpolyposis colorectal cancer (CRC), with the primary goal to develop consensus for the optimal management of this disease. A second goal was to identify areas of research with the potential to advance the clinical management of Lynch syndrome. The perspectives and recommendations from the workshop are meant to be a platform for discussion and deliberation. The Workshop was organized by Moshe Shike (Memorial Sloan Kettering Cancer Center, New York) and sponsored by The Colon Cancer Foundation. More details of each presentation are available in an on-line supplement.

Methylation in p14ARF is Frequently Observed in Colorectal Cancer with Low-level Microsatellite Instability

Colorectal cancer (CRC) can be classified as high-level microsatellite instability (MSI-H), low-level MSI (MSI-L) and microsatellite stable (MSS) depending on levels of MSI. MSI-H CRC relies on a distinct molecular pathway due to the mismatch repair (MMR) deficiency and shows methylation in multiple gene promoters. The genetic pathway leading to MSI-L is unknown, although higher levels of promoter methylation are observed in this group compared with MSS CRCs. This study explored how promoter methylation affects MSI phenotype, by analysing the methylation status of eight CRC-related promoters, MSI phenotype and KRAS/BRAF mutations in a series of 234 CRCs. Promoter methylation of p14(ARF) was significantly related to MSI-L CRC with KRAS mutation. The MSI-H phenotype was related to methylation of MLH1 as expected, while the MSS phenotype was related to methylation of p16(INK4a) and O(6)-methylguanine-DNA methyltransferase, although this was not statistically significant. Thus, promoter methylation of p14(ARF) could be a significant alteration leading to CRC with MSI-L.

HLA‐DR expression is associated with better prognosis in sporadic Australian clinicopathological Stage C colorectal cancers

Predicting patient outcome for colorectal carcinoma (CRC) with lymph node but not distant metastases remains challenging. Various prognostic markers have been identified including microsatellite instability (MSI) and possibly expression of the MHC Class II protein, HLA-DR. About 15% of sporadic CRC exhibits MSI associated with methylation of the DNA mismatch repair gene hMLH1 promoter. In addition, a significant proportion of unselected CRC demonstrates expression of HLA-DR. We sought to examine the relationship between HLA-DR expression, MSI status and prognosis in sporadic Australian Clinicopathological (ACP) Stage C CRC. Two hundred seventy consecutive patients with sporadic ACP Stage C CRC were treated at Concord Repatriation General Hospital between 1986 and 1992. None of these patients received adjuvant chemotherapy and all were followed for a minimum of 5 years or until death. DNA was extracted from paraffin sections and MSI status determined by PCR. HLA-DR expression was determined immunohistochemically using an antibody against the HLA-DR alpha chain. MSI status could be assigned in 235 cases: 176 CRCs (74.9%) were microsatellite stable, whereas 23 (9.8%) had high levels of MSI (MSI-H) and 36 (15.3%) had low levels of MSI (MSI-L). HLA-DR expression by CRC cells was seen in 148 (60.1%) cases and correlated with the presence of tumor-infiltrating lymphocytes (p = 0.0005) and peritumoral lymphocytes (p = 0.003), but not other clinicopathological features or MSI status. HLA-DR-positive CRCs were strongly associated with better patient outcome (p < 0.0001).

Sporadic colorectal carcinomas with low‐level microsatellite instability in Korea: Do they form a distinct subgroup with distinguished clinicopathological features?

The biologic significance of low-level microsatellite instability (MSI) in colorectal cancers (CRCs) remains unclear. This study aimed to elucidate whether sporadic MSI-low CRCs in Korea displayed distinguished clinicopathological characteristics from microsatellite stable (MSS) and MSI-high CRCs. We consecutively enrolled 657 patients who underwent their first surgical resections for stage I-IV sporadic CRCs and compared their clinicopathological features and prognosis after resection according to MSI status (574 MSS, 30 MSI-low and 53 MSI-high CRCs). When compared with MSS CRCs, MSI-low CRCs showed significantly more frequent association with poorly differentiated histology, mucinous carcinoma, and large tumour size. In addition, MSI-low CRCs demonstrated significantly less frequent lymph node metastasis and advanced tumour stage than MSS CRCs. When compared with MSI-high CRCs, MSI-low CRCs were significantly more frequently located in distal colon. Three-year overall and disease-free survival rates of MSS, MSI-low and MSI-high CRCs were 83.5%, 90.0% and 91.7% and 82.0%, 89.1% and 87.5%, respectively and neither demonstrated significant difference between three groups. These results indicated that sporadic MSI-low CRCs in Korea displayed distinguished clinicopathological features and might form a distinct subgroup especially from MSS CRCs. Further large studies are required to evaluate the impact of MSI-low status on prognosis.

Analysis of genomic instability in adult-onset celiac disease patients by microsatellite instability and loss of heterozygosis

Malignant complications of celiac disease (CD) include carcinomas and lymphomas. The genetic basis behind cancer development in CD is not known, but acquisition of genetic abnormalities and genomic instability has been involved. The aim of this study was to explore molecular characteristics of genomic instability in CD patients by analyzing microsatellite instability (MSI) and loss of heterozygosis (LOH) with carefully selected microsatellites. We genotyped small bowel biopsies and peripheral blood samples from 20 untreated CD patients using five microsatellites related to MMR genes (panel A), and five repeats associated with tumor suppressor genes, chromosome instability, inflammation, and cancer (panel B). Genomic instability was found in seven out of 20 (35%) cases at: D5S107, D18S58, GSTP, TP53 or DCC, being TP53 the most frequently affected (five out of seven cases; 71%). Microsatellite alterations were significantly found using panel B markers (P=0.04). No cases with high frequency of MSI and replication error phenotype were detected. Only one case displayed MSI-L alone. Three patients exhibited LOH and three other cases showed LOH with low level of MSI, being classified as having chromosome instability phenotype. Two novel observations were found in this study: first, the finding that non-neoplastic cells from a group of untreated CD patients present genomic instability at nucleotide level; and second, the advantage to use carefully selected microsatellites to identify celiac patients with molecular instability. Our data support the existence of chromosome instability phenotype in CD, suggesting that stable and unstable patients are genomically distinct subtypes that may follow a different evolution.

Most low‐level microsatellite instability in colorectal cancers can be explained without an elevated slippage rate

Many cancers show a low level of microsatellite slippage and are labelled MSI-L (microsatellite instability--low). However, it is unclear whether this slippage can be attributed to some underlying genetic change that results in a mutator phenotype, analogous to mismatch repair deficiency in MSI-H cancers, or whether the apparent instability is the result of relatively frequent normal somatic slippage. Here, we have used a mathematical model of microsatellite slippage during cancer growth to estimate the degree of microsatellite slippage expected in a cancer due to normal somatic slippage. We compared the model to the slippage observed in 42 non-MSI-H cancers that were macro-dissected into four distinct regions and genotyped at N = 9 microsatellite loci. When the slippage rate was set at mu = 10(-5) per locus per division, ten cancers showed a level of slippage in at least one region that was too severe to be expected from normal somatic slippage alone, suggesting that these cancers had acquired MSI-L. Only one of these ten cancers had putative MSI-L in all four regions. When we considered a slightly higher slippage rate of mu = 5 x 10(-5), none of the cancers showed a degree of slippage that could not be reasonably explained by normal somatic slippage. Counting the number of 'unstable' loci was a poor indicator of putative MSI-L status. We conclude that most low-level microsatellite instability in colorectal cancers can be explained without requiring an elevated slippage rate during neoplastic development, and hence there is little evidence for a discrete MSI-L group of cancers. Putative MSI-L status is indicated by the presence of at least one locus that has multiple alleles that differ by at least five motif repeats from the germline. If an underlying genetic change does cause MSI-L, it appears to be a relatively uncommon event that occurs late in oncogenesis.

A Prospective, Multicenter, Population-Based Study of BRAF Mutational Analysis for Lynch Syndrome Screening

Mismatch repair (MMR) deficiencies are the hallmark of tumors arising in Lynch syndrome, however, in approximately 15% of sporadic colorectal cancers (CRC) these deficiencies most often are associated with somatic methylation of the MMR gene MLH1. Recently, an oncogenic mutation in the BRAF gene has been involved in sporadic CRC showing MMR deficiencies as a result of MLH1 promoter methylation. The aim of this study was to evaluate the contribution of BRAF V600E mutation analysis in the identification of MSH2/MLH1 gene mutation carriers in newly diagnosed CRC patients. BRAF V600E mutation was analyzed in CRC patients with MMR deficiencies (microsatellite instability and/or lack of MLH1/MSH2 protein expression) in the EPICOLON population-based study. The effectiveness and efficiency of different strategies were evaluated with respect to the presence of MSH2/MLH1 germline mutations. MMR deficiencies were detected in 119 of the 1222 CRC patients with tumors showing either microsatellite instability (n = 111) or loss of protein expression (n = 81). BRAF mutation was detected in 22 (18.5%) of the patients. None of the patients with unambiguous germline mutation had BRAF mutation. Regardless of the strategy used to identify MSH2/MLH1 gene carriers, the introduction of BRAF analysis in these patients slightly improves their effectiveness. The introduction of BRAF mutation analysis as a step before germline genetic testing in patients with MMR deficiencies achieved a significant reduction in costs per mutation detected. Detection of BRAF V600E mutation could simplify and improve the cost effectiveness of genetic testing for hereditary nonpolyposis colorectal cancer, especially in patients whose family history is incomplete or unknown.

Microsatellite instability and clinicopathological features in esophageal squamous cell cancer

Since multiple genetic alterations are involved in the molecular pathogenesis of esophageal squamous cell cancer (ESCC), the role of microsatellite instability (MSI) in its carcinogenesis is not well defined. The reported frequency of MSI in ESCC ranges from 2 to 66.7% but the majority of the results are derived from relatively small studies. Therefore, we carried out a precise MSI analysis on a large number of ESCC samples to clarify the significance of MSI in the ESCC tumorigenesis. The MSI status of the DNA extracted from 62 ESCC samples and 62 counterpart-normal esophageal epitheliums were studied with five NCI panel markers and ten microsatellite markers located in 17q24-25. Forty-four paraffin-embedded samples and 18 frozen samples from the ESCC patients who underwent surgery were studied. The MSI status was classified as MSS (microsatellite stable), MSI-L (low-level MSI; <30% of markers examined showed instability) and MSI-H (high-level MSI; >30% of markers reported instability). Among the 62 ESCC cases analyzed by the 15 microsatellite markers, 38 out of 62 cases (61.3%) showed MSS, 19 out of 62 cases (30.6%) showed MSI-L and 5 out of 62 cases (8.1%) showed MSI-H. Although the MSI status was not associated with the status of lymph node metastasis or a histological type of cancer, the depth of cancer invasion was significantly associated with the frequency of MSS status and the levels of MSI-L were inversely correlated with the depth of invasion (T1/T2 vs. T3/4; P=0.0007). However, MSI status was not associated with the prognosis of the ESCC patients. This is the first large scale MSI analysis of the ESCC in comparison with the clinicopathological features. Relatively high frequency of MSI-L was observed in ESCC and the frequency of MSI-L was inversely correlated with the depth of invasion.