High-resolution Fluorescent Microsatellite Analysis Research Articles

Low-frequency microsatellite instability in genomic di-nucleotide sequences correlates with lymphatic invasion and poor prognosis in gastric cancer.

The clinical significance of low-frequency microsatellite instability (MSI-L) in gastric cancer (GC) has not been well established. The aim of this study was to evaluate the clinicopathological features of MSI-L in GC. We investigated microsatellite instability (MSI) in 5 di-nucleotide repeat sequences in 210 unselected GC patients. High-resolution fluorescent microsatellite analysis assay was utilized to detect MSI. Clinicopathological variables were compared among groups with different microsatellite statuses. The overall survival (OS) was analyzed by Kaplan-Meier method. Multivariable analysis was performed to identify prognostic factors and variables correlated with lymph node metastasis. High-frequency microsatellite instability (MSI-H), MSI-L, and microsatellite stable were identified, respectively, in 10.5, 10.0, and 79.5% of unselected GC cases. Tumors with MSI-H were less invasive, and these patients showed a better OS. MSI-L was correlated with more advanced tumor Node Metastasis stage, and more frequent lymph node metastasis. The unfavorable prognosis predicted by MSI-L was ascribed to its correlation with lymphatic invasion. MSI-L characterized by di-nucleotide markers represents a distinct subcategory of GC with aggressive clinicopathological features, which are particularly affiliated to lymphatic system and correlated with a poor prognosis. MSI-L could be beneficial for predicting the clinical outcome of GC.

LKB1 Mutations Frequently Detected in Mucinous Bronchioloalveolar Carcinoma

LKB1 mutations are common in patients with Peutz-Jeghers syndrome, which is characterized by mucocutaneous pigmentation, intestinal polyps and a high incidence of cancers at variable sites. This study investigated the status of the LKB1 gene in mucinous bronchioloalveolar carcinoma with or without Peutz-Jeghers syndrome. Three mucinous bronchioloalveolar carcinoma tumors from two Peutz-Jeghers syndrome patients and seven tumors from sporadic mucinous bronchioloalveolar carcinoma patients were collected by surgery between 2002 and 2008, and high molecular weight genomic DNA was extracted from them. The nucleotide sequences in exons 1-9 of LKB1 were determined by genomic polymerase chain reaction-direct sequencing. The loss of heterozygosity was analyzed by high-resolution fluorescent microsatellite analysis using two microsatellite markers that encompass the LKB1 locus, D19S886 and D19S565. The mutations of KRAS, EGFR and p53 were also evaluated. The germline mutation of LKB1 in the Peutz-Jeghers syndrome patients was identified as G215D by analyzing genomic DNA from normal lung tissue specimens. Furthermore, two of the three mucinous bronchioloalveolar carcinomas from these Peutz-Jeghers syndrome patients exhibited additional somatic mutations. On the other hand, four of seven sporadic 'non-Peutz-Jeghers syndrome' mucinous bronchioloalveolar carcinomas had LKB1 mutations. Loss of heterozygosity analyses revealed allelic loss in two tumors with LKB1 mutations. As a result, 70% of the mucinous bronchioloalveolar carcinomas exhibited LKB1 mutations. KRAS, EGFR and p53 mutations were mutually exclusive and observed in four, two and one tumors, respectively. Among them, five mutations occurred concomitantly with LKB1 mutations. The relatively high frequency of LKB1 mutations in mucinous bronchioloalveolar carcinoma patients may therefore suggest its involvement in lung carcinogenesis, at least in mucinous bronchioloalveolar carcinoma.

LKB1 mutations in mucinous bronchioloalveolar carcinoma occurring in Peutz-Jeghers syndrome patients

11047 Background: Mutations in the gene encoding Liver Kinase B1, LKB1, are common in patients with Peutz-Jeghers syndrome (PJS), which is characterized by mucocutaneous pigmentation, intestinal polyps and a high incidence of cancers at variable sites (colorectal, gynecological, breast, pancreas, and lung). Although tumors occurring in PJS patients are known to contain mucin-rich conmponents, mucinous bronchioloalveolar carcinomas (mBACs) arising from the PJS background have only rarely been reported. Here we report two mBAC patients with PJS. We further explored the LKB1 gene in these two patients and, in addition, eight sporadic mBAC patients. Methods: Frozen tissue specimens were collected from ten mBAC patients who underwent surgery in our department from 2002 to 2008, and high molecular weight genomic DNA was extracted from them and stocked in the bio-bank. Written informed consent was obtained from each patient, and ethical approval was obtained from the IRB. The nucleotide sequence of LKB1 (EX01–09) was determined by genomic PCR-direct sequencing. Loss of heterozygosity (LOH) was analyzed by high resolution fluorescent microsatellite analysis (HRFMA) using two microsatellite markers that encompass the LKB1 locus, D19S886 and D19S565. Results: Among 11 tumors derived from the 10 patients, 9 distinct LKB1 mutations were found in 7 tumors (4 G:C to A:T transitions; 3 G:C to C:G transversions; 2 single nucleotide insetion/deletion). All of three tumors obtained from the two PJS patients harbored a same sequence alteration. Although LOH was not observed in these tumors, independent sequence alterations were found in two of the three tumors, which may suggest biallelic inactivation of LKB1 in tumors occurred in the PJS patients. Conclusions: The relatively high frequency of LKB1 mutation in mBAC patients may suggest its implication in lung carcinogenesis, at least in mBAC, and its potential as a therapeutic target. [Table: see text] No significant financial relationships to disclose.

Expression of mismatch repair proteins, hMLH1/hMSH2, in non‐small cell lung cancer tissues and its clinical significance

hMLH1 and hMSH2 have been implicated to be involved in the DNA mismatch repair (MMR) system. The purpose of this study is to investigate the expression of hMLH1 and hMSH2 DNA MMR proteins in non-small cell lung cancer (NSCLC) tissue and to elucidate their clinical significance. The hMLH1 and hMSH2 protein expression was evaluated by immunohistochemistry for a consecutive series of 113 NSCLC patients. The expressions of each protein were examined for an association with the clinicopathological variables, including genetic alterations analyzed by high resolution fluorescent microsatellite analysis. Regarding the hMLH1 expression, the MSI-positive patients showed significantly lower scores than the MSI-negative patients. For hMSH2 expression, the patients with a 20 or higher pack-year index (PYI) showed significantly higher scores than the patients with a PYI less than 20. The expression status of proteins did not affect both the disease free and overall survival of the patients. No significant correlation was observed among the scores for the proteins. The expressions of hMLH1 and hMSH2 are independently regulated and play different roles in NSCLC. The genetic instability is possibly due to the reduced expression of hMLH1 protein, and hMSH2 expression is associated with smoking status.

High-resolution fluorescent analysis of microsatellite instability in gastric cancer

Microsatellite instability (MSI) is associated with various human malignancies and regarded as reflecting cellular deficiency in DNA mismatch repair (MMR). Analysis of MSI has been prevalent in the field of oncology, and numerous data have accumulated in the literature. It has been reported that the MSI+ phenotype is relatively frequent in gastric cancer. The reported frequencies of MSI+ gastric tumors, however, are diverse. To determine the frequencies of the MSI+ phenotype and defective MMR in gastric cancer, we examined tumors derived from 167 patients with sporadic gastric cancer, using our unique fluorescent technique, 'high-resolution fluorescent microsatellite analysis'. High-resolution fluorescent microsatellite analysis allowed us the unequivocal designation of MSI. The frequencies of MSI-H and MSI-L were 11 and 9.6%, respectively. In addition to the distinction based on the frequency of microsatellite changes, MSI was classifiable into two distinct categories, type A and type B, according to the mode of length changes in the dinucleotide microsatellites. Type A and type B MSI were observed in 14 and 6.6%, respectively. The overall frequency of MSI was 21%. Intriguingly, MSI did not correlate with any of commonly used clinicopathological variables. In addition, neither MSI-H nor MSI-L correlated with family history of malignancies or patient history of multiple cancers. Instead, type B MSI was significantly more frequent in patients with family history of gastric cancer. Type A MSI appeared to occur more frequently in tumors of patients with a history of double cancer, which, however, was not statistically significant. In gastric cancer, contribution of defective MMR to the risk of multiple cancer or familial predisposition appears more limited than has been expected. The relationship between MSI and high risk of cancer may have been oversimplified, at least in gastric cancer.

Coexistence of the loss of heterozygosity at the PTEN locus and HER2 overexpression enhances the Akt activity thus leading to a negative progesterone receptor expression in breast carcinoma

Serine/threonine kinase Akt/PKB is known to regulate divergent cellular processes, including apoptosis, proliferation, differentiation, and metabolism. Akt is activated by a variety of stimuli, through such growth factor receptors as HER2, in phosphoinositide-3-OH kinase (PI3K)-dependent manner. A loss of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) function also activates Akt. It has recently been shown that Akt activation is associated with a worse outcome among endocrine treated breast cancer patients and that it also inhibits the progesterone receptor (PR) expression via the PI3K/Akt pathway in breast cancer cells. Therefore, the PI3K/Akt signaling pathway has recently attracted considerable attention as a new target for effective therapeutic strategies. In the present study, we investigated the relationship between Akt activation and either HER2 overexpression or PTEN gene alteration, as well as the PR expression. We analyzed the incidence of LOH at the PTEN locus in 138 breast cancer patients, using our new system for microsatellite analysis, called high-resolution fluorescent microsatellite analysis (HRFMA). We showed Akt activation to significantly correlate with HER2 overexpression or LOH at the PTEN gene locus while inversely correlating with the PR expression. In addition, when LOH at the PTEN gene locus and HER2 overexpression occurred simultaneously, the incidence of Akt activation and reduced PR expression was significant. The association between Akt activation and PR negative expression was observed even in the ER-positive cases. Our results suggest that simultaneous PTEN LOH and HER2 overexpression enhances Akt activation and may thus lead to a negative PR expression.

Microsatellite instability and proliferating activity in sinonasal carcinoma: Molecular genetic and immunohistochemical comparison with oral squamous cell carcinoma

Sinonasal carcinomas arise from the respiratory epithelium that lines the nasal and paranasal cavities, and are histologically composed of either squamous or cylindrical cell carcinoma. However, molecular analysis with the purpose of distinguishing sinonasal carcinomas from other head and neck squamous cell carcinomas (HNSCCs), which arise from squamous epithelium, has been limited. Moreover, a wide range of frequency of microsatellite instability (MSI) in HNSCC has been reported. Using high-resolution fluorescent microsatellite analysis (HFRMA), we studied microsatellite alterations in 34 patients with sinonasal carcinoma. As a control, 24 oral squamous cell carcinomas were used. MSI was detected in 14 patients with sinonasal carcinoma (41%), but not in any with oral squamous cell carcinoma (p=0.002). Furthermore, in sinonasal carcinoma, 11 out of 17 (65%) T1-T3 sinonasal carcinomas demonstrated MSI, whereas only 3 out of 15 (20%) T4 tumors demonstrated MSI. Immunohistochemically, sinonasal carcinoma showed a higher MIB-1-labeling index and more frequently showed cytokeratin 18 expression when compared with oral squamous cell carcinoma. These findings suggest that sinonasal carcinoma and HNSCC have quite different molecular backgrounds regarding carcinogenesis, and the role of MSI is relatively minor in cases of advanced sinonasal carcinoma.

Microsatellite instability and hMLH1 and hMSH2 expression analysis in soft tissue sarcomas.

Alterations of the size of microsatellite DNA sequences, namely microsatellite instability (MSI), have been demonstrated in some types of malignancies. We analyzed the MSI of five microsatellite markers in 40 cases of soft tissue sarcoma (STS) using high resolution fluorescent microsatellite analysis. In addition, we examined the expression of hMLH1 and hMSH2 proteins of DNA mismatch repair (MMR) genes by immunohistochemistry, and promoter methylation of the hMLH1 gene by methylation-specific PCR (MSP). MSI was recognized in 10 of 40 STS cases (25%), which consisted of 2 MSH-high (MSI-H) tumors and 8 MSI-low (MSI-L) tumors. A loss of hMLH1 expression was recognized in 7 of 40 STS cases (18%), and loss of hMSH2 expression was recognized in 3 of 40 STS cases (8%). One case showed a loss of both hMLH1 and hMSH2 expression. Promoter hypermethylation of the hMLH1 gene was detected in only 3 of 40 STS cases (8%). Of 10 cases with MSI, 5 (50%) showed a loss of hMLH1 and/or hMSH2 expression. There was a statistically significant correlation between MSI-positive tumors and the loss of hMLH1 and/or hMSH2 expression (p=0.0286). Although the frequency of MSI (25%) or a loss of hMLH1 and/or hMSH2 expression (23%) was relatively low in STS cases, a loss of hMLH1 and/or hMSH2 was recognized in 5 out of 10 MSI-positive cases (50%). These findings suggest that the inactivation of MMR gene expression might be the cause of MSI in STS cases.

Frequent Loss of Heterozygosity but Rare Microsatellite Instability in Oesophageal Cancer in Japanese and Chinese Patients

Reported frequencies for microsatellite instability (MSI) in oesophageal cancer differ widely in the literature, perhaps due to the high incidence of loss of heterozygosity (LOH) in this cancer. Using high-resolution fluorescent microsatellite analysis (HRFMA), we analysed microsatellite alterations in detail in 50 Japanese and 50 Chinese patients with squamous cell carcinoma in the oesophagus. In HRFMA, several devices have been developed to improve the detection characteristics, reproducibility of polymerase chain reaction and the migration accuracy of electrophoresis. All the alterations observed were separable into MSI, LOH and alterations ambiguous for both. MSI was rare in these panels of oesophageal carcinomas. The frequencies of MSI in the Japanese and Chinese subjects were 8 and 4%, respectively. All the alterations were mild (within 2 base pairs) and were observed in a limited number of markers. More drastic types of MSI, such as those typical in colorectal cancer, were not observed. On the other hand, the incidence of LOH was high, reaching 50% for the Japanese and 70% for the Chinese subjects. In many of these cases, LOH was observed in multiple microsatellite markers. The frequency of LOH in each marker was not apparently biased. Although in many cases MSI and LOH were clearly distinguished with use of the sensitive and quantitative fluorescent assay, theoretically indistinguishable patterns were noted in some cases. In conclusion, MSI is rare and LOH predominates in squamous cell carcinoma in the oesophagus.

Features of microsatellite instability in gastric and colorectal cancer: an assessment by High Resolution Fluorescent Microsatellite Analysis

Features of microsatellite instability in gastric and colorectal cancer: an assessment by High Resolution Fluorescent Microsatellite Analysis

Cell kinetics and genetic instabilities in differentiated type early gastric cancers with different mucin phenotype

To clarify the biological impact and molecular pathogenesis of cellular phenotype in differentiated-type gastric cancers (DGCs), we investigated cell kinetics and genetic instabilities in early stage of DGCs. A total of 43 early gastric cancers (EGCs) were studied. EGCs were divided into 3 phenotypic categories: gastric (G type, n = 11), ordinary (O type, n = 20), and complete intestinal (CI type, n = 12) based on the combination of HGM, ConA, MUC2, and CD10. Proliferative index (PI), apoptotic index (AI), and p53 overexpression were investigated by immunohistochemical staining with anti-Ki-67, the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling method, and p53 antibody, respectively. Using a high-resolution fluorescent microsatellite analysis system, microsatellite instability (MSI) and loss of heterozygosity (LOH) were examined. Frameshift mutation analysis of transforming growth factor-β type II receptor (TGF-βRII) and bcl-2–associated X (BAX) in cancers with MSI was also performed. The mean AI/PI ratio values were 0.04 for G-type, 0.10 for O-type, and 0.13 for CI-type cancers—significantly lower in G type than in O and CI types (P = 0.02 and P = 0.001, respectively). No difference in the incidence of MSI and LOH was seen among the 3 cellular phenotypes. However, the major pattern of MSI, which showed drastic and widely dispersed changes and is related to an increased risk for cancer, was significantly higher in G and O types than in CI type (P <0.005). No frame shift mutations of TGF-βRII or BAX were found in CI-type cancers. These results indicate that G-type cancers are likely to show more aggressive behaviors than CI-type cancers, and that O-type cancers show the intermediate characteristics of both types. However, the molecular pathogenesis of each phenotypic cancer is not associated with microsatellite alterations. HUM PATHOL 34:32-40. Copyright 2003, Elsevier Science (USA). All rights reserved.

Frequency of Microsatellite Instability inBreast Cancer Determined by High-Resolution Fluorescent Microsatellite Analysis

In breast cancer, the rates of positivity for microsatellite instability (MSI), vary greatly in the literature. Using high-resolution fluorescent microsatellite analysis (HFRMA), we studied microsatellite alterations in 75 patients with sporadic breast cancer. In this system, several devices were prepared to improve reproducibility of polymerase chain reaction products, migration accuracy of electrophoresis, and characteristics of the detection system. Precise and objective analyses of microsatellite alterations are made feasible using HRFMA. Seven of the 75 cases were judged to be positive for MSI, the rate of positivity being 9.3%. This rate is relatively low compared to the data in the literature. All the microsatellite changes observed in this system can be classified into two types: type A with relatively small changes in microsatellite sequences observed in limited loci and type B with drastic and widely dispersed changes. The former was thought to be connected to abnormal activity in DNA mismatch repair (MMR). Among the 7 cases, 6 (8.0%) had type A alterations, which means that the tumors may have an abnormal MMR activity. Application of precise and objective systems for microsatellite analysis is expected to be clinically useful to detect patients at high risk for cancers.

Patient survival and microsatellite instability in gliomas by high-resolution fluorescent analysis.

Deficient repair of nucleotide mismatches in the genome is considered a major factor in tumorigenesis. Such deficiency is evidenced by alterations in dinucleotide repeats of microsatellite sequences, specifically microsatellite instability (MSI) or replication errors. We investigated the frequency of MSI in human gliomas in terms of patient outcome. Frequency of MSI was estimated by examining five loci on chromosomes 2, 5, 10, 11, and 13 in 31 gliomas using high-resolution fluorescent microsatellite analysis. MSI was found at all loci in only 2 malignant gliomas (6.5%). MSI was detected at the D10S197 locus in 3 of 11 glioblastomas (27.2%) and 4 of 8 anaplastic astrocytomas (50%), while no MSI was detected in low-grade gliomas. Among patients with anaplastic astrocytoma, the 4 with MSI at D10S197 died from local recurrence less than 18 months after surgery, while 3 of the patients without MSI survived for more than 20 months. MSI at D10S197 may be a prognostic marker for patients with anaplastic astrocytomas.

A microsatellite instability analysis in neuroblastoma based on a high resolution fluorescent microsatellite analysis

It has recently been reported that mismatch repair enzymes, which are one type of DNA repair enzymes, are the causative genes for a major group of hereditary non-polyposis colon cancers (HNPCC). Abnormalities in the mismatch repair system can be monitored by observing instability at the microsatellite loci (MSI) in cancer cells. MSI has been reported not only in tumors associated with hereditary non-polyposis colorectal cancer but also in sporadic forms of various tumors. No correlation between pediatric malignant tumors and the mismatch repair system has yet been reported. In the present study, we examined the frequency of MSI in 21 neuroblastomas, which are the most common solid tumors in childhood, using a high resolution fluorescent microsatellite analysis. MSI on five microsatellite loci was detected in none of the 21 samples. Other mechanisms independent of mismatch repair deficiency may thus play a role in both tumorigenesis and the development of neuroblastoma.

Precise assessment of microsatellite instability using high resolution fluorescent microsatellite analysis.

The instability of microsatellite sequences dispersed in the genome has been linked to a deficiency in cellular mismatch repair. This phenotype has been frequently observed in various human neoplasms and is regarded as a major factor in tumorigenesis. To demonstrate alterations in microsatellite sequences, polymerase chain reaction (PCR) and electrophoretic analysis are techniques often used. However, the electrophoretic profiles of PCR-amplified microsatellite sequences have not been well characterized. Moreover, the conventional method using autoradiography has critical problems in detection characteristics and migration accuracy. We made use of fluorescence-labeled PCR and laser scanning with linear detection characteristics, so as to detect bands quantitatively. Next, we characterized Taq polymerase-dependent modification of the amplified microsatellite sequences, using artificially synthesized microsatellite alleles and we optimized the electrophoretic profiles by enzymatic modification with T4 DNA polymerase. We developed a dual fluorescence co-electrophoresis system, in which both samples derived from cancer and normal tissues are electrophoresed in the same lane, in order to minimize migration errors. These improvements remarkably facilitate precise and objective assessments of microsatellite instability. Analyzing many positive cases in cell lines and tissue specimens, we classified all the patterns of microsatellite alteration and set up new criteria for assessing microsatellite instability.