Simultaneous analysis of microsatellite instability and loss of heterozygosity by capillary electrophoresis with a homemade kit

Microsatellite instability (MSI) and loss of heterozygosity (LOH), the alteration in length and strength of short tandem repeat sequences are an important molecular characteristic of many human tumors. MSI and LOH analysis has become an attractive method for diagnostic and tumor research purposes. A method for the simultaneous analysis of MSI and LOH at the five microsatellite loci (BAT-26, D17S261, D3S1283, D2S123 and D3S1611) was developed employing a cheap homemade kit to replace the expensive commercial kit on ABI 310 capillary genetic analyzer. After studying the effect of temperature and urea denaturant on microsatellite analysis, 8 mol/L urea and 60 degrees C were selected for assessing accurately fragment size of microsatellite alleles. Based on this method, 52 sporadic gastric cancers were screened, and MSI and LOH, at least one locus was observed in 15 of 52 (28.8%) patients. Moreover, it is found that a statistically significant association exists between MSI and LOH and tumor-differentiated level.     

Loss of heterozygosity and microsatellite instability in tumor-associated stromal cells and tumor epithelium of prostate cancer

In the past decade, intense studies of the tumor microenvironment yielded ample data testifying to the critical role of stroma in carcinogenesis. Genetic lesions accumulate not only in the tumor epithelium, but also in tumor-associated fibroblasts. The epithelial and stromal components of prostate cancer (PC) and prostatic intraepithelial neoplasia (PIN) were isolated by laser capture microdissection and subjected to microsatellite analysis of chromosome regions 8p22, 13q14, and 16q23. The frequency of allele alterations in the epithelium was 48% for 8p22, 72% for 13q14, and 37% for 16q23. Slightly higher frequencies of the loss of heterozygosity (LOH) and microsatellite instability in these loci were observed in tumor-associated stroma. Molecular alterations were also found in both epithelial (16–27%) and stromal (8–22%) components in PIN. LOH on chromosomes 16 and 13 in the epithelium was significantly associated with the Gleason score, PC stage, and metastasis into regional lymph nodes. Thus, multiple genetic aberrations occur in the stromal component of PC as frequently as in the tumor epithelium.     

Potential use of loss of heterozygosity in pleural effusions of breast cancer metastases using the microsatellite marker of the 16q22.1 region of the CDH1 gene

OBJECTIVE: To evaluate the presence of allelic loss in 16q22.1, including the locus of E-cadherin, in pleural effusions in breast cancer patients. STUDY DESIGN: Molecular analysis of DNA was performed using a DNA extraction kit (NucleoSpin, Macherey-Nagel, Germany). Loss of heterozygosity (LOH) in primary tumors and pleural effusions was analyzed using a microsatellite marker of the CDH1 gene, D16S265, described in previous studies. LOH was evaluated by radioactive polymerase chain reaction assay in 17 samples of pleural effusions and breast tissues (primary tumors and nonneoplastic adjacent tissue) from breast cancer patients: 7 positive for neoplastic cells, 6 suspected and 4 cases without evidence of neoplastic cells in the effusions. RESULTS: Thirteen cases (76%) were informative. LOH was detected in 5 cases (38.5%). In 3 of them LOH was detected only in the cytologic sample, and in 2 of them LOH was detected in the primary tumor and cytologic sample. CONCLUSION: Results show that LOH in the CDH1 gene can identify tumor cells in pleural effusions when morphologic analysis is difficult.     

Analysis of genome instability in breast cancer

Breast cancer is a heterogeneous disease, previously associated with genomic instability. Our aim was to analyze microsatellite markers in order 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 107 breast carcinomas at twelve microsatellite loci. Some of the markers were selected because of their relation to steroid hormone metabolism, which seems to be related to sporadic breast cancer risk. D5S346 and D17S250 markers showed a statistically significant frequency of MSI. LOH in D3S1611, D17S250, AR and ER-β were associated with some parameters of worse prognosis. Marker group analysis showed that CYP19, AR and ER-β were related to histological grade III, ER-negative and PR-negative cases. Our results suggest that marker group analysis may be preferred to the single marker strategy, being predictive of worst prognosis when single markers are unable to provide such information. A further evaluation of steroid metabolism genes and their association with low penetrance genes in breast cancer may be useful.     

Clinicopathologic features in colorectal cancer patients with microsatellite instability

The microsatellite instability (MSI) mutational pathway is critical to carcinogenesis in a small but significant proportion of colorectal cancers. While MSI is identified in most cancers in individuals with hereditary non-polyposis colorectal cancer, the majority of MSI tumors are found in individuals with sporadic disease. Colorectal cancers arising as a result of MSI have distinct clinicopathologic features distinguishing them from those with microsatellite stability. MSI colorectal cancers affect a larger percentage of women, are usually localized proximal to the splenic flexure, and have a higher incidence of synchronous and metachronous tumors. They are associated with a mucinous histology, tumor-infiltrating lymphocytes, a Crohn's-like inflammatory response, and a higher grade but lower stage. Overall survival is better in individuals with MSI. The benefit of chemotherapy in MSI colorectal cancers, with and without lymph node metastases, remains unclear.     

Development of a yeast-based assay system for monitoring microsatellite instability

Simple sequence repeats (microsatellites) are found in all eukaryotic genomes. Instabilities within these sequences have been associated with several human disorders including Huntington's chorea and myotonic dystrophy. Further studies have identified links between microsatellite instability, faulty mismatch repair and certain human cancers, in particular a form of hereditary colorectal cancer. The assay system described here consists of a congenic set of yeast strains mutated in DNA replication and mismatch repair genes and assay plasmids with which it is possible to measure differences in microsatellite stability in the range of 5-850-fold. The development of this technology will allow monitoring of environmental and dietary influences on the genomic stability in the context of human disease.     

Assessment of microsatellite instability in colorectal cancer patients from Brazil

The replication error status analysis of DNA, through microsatellite instability detection, has become an indispensable tool for hereditary non-polyposis colorectal cancer screening. This study investigated the microsatellite instability in Brazilian individuals presenting colorectal cancer. In this study, 66 patients were clinically analyzed according to Amsterdam II and Bethesda guidelines. Normal and tumour tissues were collected and analyzed for MSI degree according to molecular markers BAT25, BAT26, BAT40, APC–D5S346, D2S123, and D17S250. Eight patients (12.1%) fulfilled the Amsterdam II guidelines, and 15 (22.7%) met the Bethesda guidelines. BAT25 was the most sensitive marker (86.7%), while BAT26 was the least sensitive (66.7%). The specificity of both markers was 100%, but all of the markers must be used since the contribution of each marker to the sensitivity and specificity of the test is complementary. Proximal tumours were significantly predominant among RER⁺ patients. Conclusions: Patients with a family history of colorectal cancer with the tumour in the proximal colon must be screened to replication error status as early as possible in order to avoid the progression of the disease.     

Mitochondrial microsatellite instability in gastric cancer and gastric epithelial dysplasia as a precancerous lesion

Background: Genetic instability in gastric cancer represents a key molecular step that occurs early in the carcinogenesis process. To clarify the role of genetic instability in the progression from gastric dysplasia to gastric cancer, mitochondrial microsatellite instability (mtMSI) was studied in gastric cancer and gastric dysplasia. Methods: DNA was isolated from paired normal and tumoral tissues in 24 patients with gastric dysplasia (low grade) and 49 patients with gastric cancer. mtMSI was analyzed using eight microsatellite markers. mtMSI in gastric dysplasia was studied prospectively to elucidate the relation between mtMSI and gastric carcinogenesis. Results: mtMSI was found in 5 (10.2%) of 49 gastric cancer patients. The mtMSI phenotype was not associated with age, gender, and Helicobacter pylori infection. However, all of the mtMSI was found in intestinal-type gastric cancer (20.8%, p = 0.02). In gastric dysplasia, mtMSI was detected in 3 (12.5%) of 24 patients with gastric dysplasia. mtMSI-positive gastric dysplasia showed a poor prognosis statistically compared to mtMSI negative through progression to high-grade dysplasia or gastric cancer. Conclusions: These data suggest that mtMSI may be an early and important event in the progression of gastric carcinogenesis, especially in intestinal-type gastric cancer.     

Thymidylate synthase polymorphism and microsatellite instability: association in colorectal cancer

5-Fluorouracil (5FU) is the main drug used for the treatment of colorectal cancer (CRC) and Thymidilate Synthase (TS) is its target enzyme. TS gene has regulatory tandemly repeated sequences in its 5' and 3'untraslated region (5'-3' UTR). CRC often shows a kind of genomic instability called Microsatellite Instability (MSI) that is associated with TS levels and survival. Our data show that the genotype 2R/2R (homozygosity for 2 tandem repeat sequences in the 5'UTR) is more frequently associated with MSI+ and lower TS levels. More over we did not find any significant association between the 2R/3R (heterozygosity for 2 and 3 tandem repeat sequences in the 5'UTR) and 3R/3R (homozygosity for 3 tandem repeat sequences in the 5' UTR) genotypes with the MSI+ and MSI-, while these genotypes were associated with a higher TS expression. As a consequence we can hypothesise that patients bearing CRC with the MSI+, the 2R/2R genotype and with low TS levels could have a better prognosis and they could not be drug resistant.     

Assessment of microsatellite instability in head and neck cancer using consensus markers

Head and neck cancer is the sixth most common cancer in the world and one of the most lethal cancers. Microsatellite instability is an important characteristic of tumor cells and is observed both in presence and absence of mismatch repair gene mutations. The importance of microsatellite instability in head and neck cancer is not well established due to the lack of a consensus panel and selection of different markers, criteria and methodological variances. The main objective of this study was to investigate the performance of a consensus panel of microsatellite repeats by automated fragment analysis. Matched tumor and normal tissue samples from 99 patients were analyzed using five mononucleotide markers. Following PCR the amplified fragments were analyzed by capillary electrophoresis on an ABI 310 genetic analyzer. Microsatellite instability was observed in 26 patients. In 17 patients instability was detected at multiple loci. NR21 and BAT25 were the most frequently altered targets. These two mononucleotide markers could detect all samples displaying high-instability. In this study we describe a standardized fluorescent multiplex PCR combined with computerized analysis, which allows rapid and accurate analysis of a high number of samples and obviates the need to compare tumors with matching normal tissue.     

Chromosomal instability in the lymphocytes of breast cancer patients

Genomic instability in the tumor tissue has been correlated with tumor progression. In the present study, chromosomal aberrations (CAs) in peripheral blood lymphocytes (PBLs) of breast tumor patients were studied to assess whether chromosomal instability (CIN) in PBLs correlates with aggressiveness of breast tumor (i.e., disease stage) and has any prognostic utility. Cultured blood lymphocyte metaphases were scored for aberrations in 31 breast cancer patients and 20 healthy age and sex-matched controls. A variety of CAs, including aneuploidy, polyploidy, terminal deletions, acentric fragments, double minutes, chromatid separations, ring chromosome, marker chromosome, chromatid gaps, and breaks were seen in PBLs of the patients. The CAs in patients were higher than in controls. A comparison of the frequency of metaphases with aberrations by grouping the patients according to the stage of advancement of disease did not reveal any consistent pattern of variation in lymphocytic CIN. Neither was any specific chromosomal abnormality found to be associated with the stage of cancer. This might be indicative of the fact that cancer patients have constitutional CIN, which predisposes them to the disease, and this inherent difference in the level of genomic instability might play a role in disease progression and response to treatment.     

Chromosome-breakage genomic instability and chromothripsis in breast cancer

Background

Chromosomal breakage followed by faulty DNA repair leads to gene amplifications and deletions in cancers. However, the mere assessment of the extent of genomic changes, amplifications and deletions may reduce the complexity of genomic data observed by array comparative genomic hybridization (array CGH). We present here a novel approach to array CGH data analysis, which focuses on putative breakpoints responsible for rearrangements within the genome.

Results

We performed array comparative genomic hybridization in 29 primary tumors from high risk patients with breast cancer. The specimens were flow sorted according to ploidy to increase tumor cell purity prior to array CGH. We describe the number of chromosomal breaks as well as the patterns of breaks on individual chromosomes in each tumor. There were differences in chromosomal breakage patterns between the 3 clinical subtypes of breast cancers, although the highest density of breaks occurred at chromosome 17 in all subtypes, suggesting a particular proclivity of this chromosome for breaks. We also observed chromothripsis affecting various chromosomes in 41% of high risk breast cancers.

Conclusions

Our results provide a new insight into the genomic complexity of breast cancer. Genomic instability dependent on chromosomal breakage events is not stochastic, targeting some chromosomes clearly more than others. We report a much higher percentage of chromothripsis than described previously in other cancers and this suggests that massive genomic rearrangements occurring in a single catastrophic event may shape many breast cancer genomes.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-579) contains supplementary material, which is available to authorized users.     

Investigating loss of heterozygosity in a SCRaMbLEd yeast genome

Science China Life Sciences -     

High frequency of microsatellite instability in sporadic colorectal cancer patients in Iran

Microsatellite instability in sporadic colorectal cancer patients was assessed, and the clinicopathological associations were evaluated in northeastern Iran, which is a high-risk region for gastrointestinal malignancies. Microsatellite instability (MSI) status of tumoral tissue, compared to normal tissue, was assessed with a standard panel of MSI markers on paraffin-embedded surgically resected tissues from 67 consecutive sporadic colorectal cancer patients. Eleven of the patients were under 40 years old. Female patients were significantly younger than male patients (mean age 54.2 vs 62.1 years, P = 0.020). MSI analysis revealed 18 cases of MSI-H (26.9%), 11 MSI-L (16.4%) and 38 MSS (microsatellite stable tumors; 56.7%). While a greater proportion of patients consisted of males, 56.7 vs 43.3% females, MSI-H was more frequent in females (34.5 vs 21.5%). MSI was associated with proximal location of tumor (P = 0.003) and lower stages of tumor (P = 0.002), while MSS tumors were associated with node metastasis. MSI has a higher frequency in sporadic colorectal cancer patients, suggesting that molecular epidemiology of the genetic alterations involved in colorectal cancer carcinogenesis has a different pattern in the Iranian population, which deserves further epidemiological attention. The high frequency of MSI-H in this population suggests that we should look at microsatellite instability prior to chemotherapy to determine the most appropriate chemotherapeutic strategy in our population.     

Pooled analysis of loss of heterozygosity in breast cancer: a genome scan provides comparative evidence for multiple tumor suppressors and identifies novel candidate regions

Somatic loss of heterozygosity (LOH) has been widely reported in breast cancer as a means of identifying putative tumor-suppressor genes. However, individual studies have rarely spanned more than a single chromosome, and the varying criteria used to declare LOH complicate efforts to formally differentiate regions of consistent versus sporadic (random) loss. We report here the compilation of an extensive database from 151 published LOH studies of breast cancer, with summary data from >15,000 tumors and primary allelotypes from >4,300 tumors. Allelic loss was evaluated at 1,168 marker loci, with large variation in the density of informative observations across the genome. Using studies in which primary allelotype information was available, we employed a likelihood-based approach with a formal chromosomal instability and selection model. The approach seeks direct evidence for preferential loss at each locus compared with nearby loci, accounts for heterogeneity across studies, and enables the direct comparison of candidate regions across the genome. Striking preferential loss was observed (in descending order of significance) in specific regions of chromosomes 7q, 16q, 13q, 17p, 8p, 21q, 3p, 18q, 2q, and 19p, as well as other regions, in many cases coinciding with previously identified candidate genes or known fragile sites. Many of these observations were not possible from any single LOH study, and our results suggest that many previously reported LOH results are not systematic or reproducible. Our approach provides a comparative framework for further investigation of regions exhibiting LOH and identifies broad genomic regions for which there exist few data.     

转基因小鼠和基因突变小鼠微卫星不稳定性分析

目的探讨微卫星在转基因和基因突变小鼠中的变化,为基因修饰和遗传突变动物的遗传检测和表型分析提供理论依据和技术手段。方法根据文献报道,从GenBank中选取198个等位基因数量多、富含多态性的微卫星位点,以野生型动物为对照,对6种近交系遗传背景的转基因小鼠和5种自然基因突变的近交系小鼠进行微卫星多态性检测,选用1.5%琼脂糖凝胶电泳和STR扫描技术,比较分析微卫星不稳定性。结果共有40个微卫星位点在转基因和基因突变小鼠中表现出多态性。在基因突变小鼠中,微卫星不稳定性有55.6%(10/18)是由纯合变为杂合(Ⅰ型),有3个位点(16.6%,3/18)是纯合突变(Ⅱ型),有5个位点同时存在2种类型的突变。但是在转基因动物中,大多数的微卫星多态性为Ⅰ型突变(87.5%,28/32),只有2个位点(6.2%,2/32)是Ⅱ型突变。另外有2个位点同时存在2种类型的突变。结论基因修饰或基因突变可引起小鼠相关微卫星发生不稳定性,而且某些微卫星位点对基因改变敏感性较高。     

Four separate regions on chromosome 17 show loss of heterozygosity in familial breast carcinomas

Two genes predisposing females to autosomal dominant breast cancer are located on chromosome 17. Mutations in the p53-gene on the short arm have been shown to predispose females to early onset breast cancer in families with the rare Li-Fraumeni syndrome. Another locus on 17q (BRCA1), was found to be linked to the disease in a subset of families with breast cancer. In order to determine the involvement of tumour suppressor genes at these loci in tumour development, we studied allele losses for markers on chromosome 17 in 78 familial breast carcinomas. The analysis used six polymorphic DNA markers, three on each arm. We found support for at least four separate regions displaying allele losses on chromosome 17: the p53-region, the distal part of 17p, the BRCA1 region and the distal part of 17q. The frequency of allele losses on distal 17p (16%) is low in these familial tumours compared with the previously reported incidence in sporadic tumours (>50%), whereas the frequency of losses at the p53 locus and on 17q was similar to sporadic tumours (5%–40%). These data suggest that several regions on chromosomal 17 can harbour tumour suppressor genes involved in tumour development of familial breast cancer.