Prognostic significance of microsatellite alterations at 1p36 in cholangiocarcinoma

AIM: To investigate loss of heterozygosity (LOH) and microsatellite instability (MSI) on the chromosomal region 1p36-pter in cholangiocarcinoma (CCA) patients and determine the association between microsatellite alterations and clinicopathological parameters. METHODS: Ten polymorphic microsatellite markers were determined for LOH and MSI using GS-3000 gel scan fragment autoanalyzer. RESULTS: Sixty-eight out of 90 cases (75.6%) showed LOH in one or more loci. LOH was found most frequently at D1S199 (40.0%), D1S507 (34.6%), D1S2845 (30.5%), and D1S2734 (30.1%). MSI was found in 34 of 90 cases (37.8%) at one or more loci. Fine mapping at 1p36 showed two distinctive regions of common loss, which were D1S2845 and the 25.5-cM region between D1S507 and D1S2734, indicating the existence of putative tumor suppressor genes that is likely to play important roles in the development of CCA. Patients with LOH at D1S234 showed less lymphatic invasion (P = 0.017), whereas patients with LOH at D1S2676 exhibited more lymphatic invasion than those without (P = 0.031). LOH at D1S2845 showed a significant correlation with nerve invasion (P = 0.029). Moreover, patients who demonstrated MSI at D1S228 showed a poor prognosis (P = 0.0026). CONCLUSION: Allelic loss plays a major role in microsatellite alterations at chromosome 1p36, which may contribute to carcinogenesis and pathogenesis of liver fluke related CCA and these alterations can be used as molecular prognostic indicators for CCA patients.     

Esophageal squamous cell carcinomas with DNA replication errors (RER+) are associated with p16/pRb loss and wild-type p53

PURPOSE: Microsatellite instability (MSI) as a determinant of propensity to esophageal squamous cell carcinoma (ESCC) at seven microsatellite markers at 2p (2p15-16), 3p (3p13, 3p14.1-3, 3p25, and 3p26) and 16q (16q12.1-3) was investigated to analyze their putative role as indicators of predisposition to esophageal malignancies. METHODS: Seven microsatellite loci were amplified by polymerase chain reaction, from surgically resected tumor tissues from 30 ESCC patients from Indian population, to assess the loss of heterozygosity (LOH) and replication error repeats (RER) and to correlate these alterations with aberrations in major cell cycle regulatory proteins and histopathological parameters. RESULTS: LOH and RER analyses at these loci demonstrated moderate microsatellite alterations, suggesting the involvement of MSI in esophageal tumorigenesis in a subset of the Indian population. MSI, defined as RER in at least two or more of the loci studied, was observed in ten of 30 (33%) patients. Twenty-two of 30 patients (73%) showed LOH at one or more loci, while 17 of the 30 patients (60%) showed RER in at least one of the loci studied. RER-positive patients showed a trend towards better prognosis when compared to RER-negative patients. MSI demonstrated a significant association with concomitant loss of p16 and pRb (p16-/pRb- phenotype) (P=0.046). Interestingly, we observed an inverse correlation between MSI and p53 mutations (P=0.03) suggesting that MSI may provide a p53-independent pathway for esophageal tumorigenesis in RER+ patients. MSI showed a trend towards longer survival and absence of distant organ metastasis (P=0.06). CONCLUSIONS: The present study demonstrates the probable role of MSI in esophageal squamous cell carcinoma in the Indian population. Instability associated with the repetitive sequences--the revealing marks of loss of DNA replication fidelity may serve as an indicator of predisposition to esophageal cancer.     

老年人食管鳞状上皮化生和不典型增生及腺癌序列微卫星改变

目的评估老年人食管鳞状上皮和化生-不典型增生-腺癌的微卫星变化。方法应用稀释性聚合酶链反应（PCR）方法检测存档手术切除的食管癌标本中的D2S123、D3S1616、D3S1300、BATRII、D5S346、D17S787和D18S61位点微卫星的变化。结果在非稀释DNA中，17例食管鳞状细胞癌和12例腺癌微卫星不稳定性（MSI）的频率分别是52．9％（9例）和41．7％（5例），杂合性丢失（LOH）的频率分别是23．5％（4例）和16．7％（2例），两者差异均无统计学意义（P〉0．05）。在8例食管鳞状上皮和化生-不典型增生-腺癌组织稀释DNA中，MSI和LOH频繁出现，与其非稀释DNA的结果比较，差异均有统计学意义（P〈0．05）。结论MSI和LOH在上述组织中普遍存在，它们可能是食管腺癌发生、发展的早期事件。     

Microsatellite alterations in phenotypically normal esophageal squamous epithelium and metaplasia-dysplasia-adenocarcinoma sequence

AIM： To investigate the microsatellite alterations in phenotypically normal esophageal squamous epithelium and metaplasia-dysplasia-adenocarcinoma sequence.
METHODS： Forty-one specimens were obtained from esophageal cancer （EC） patients. Histopathological assessment identified 23 squamous cell carcinomas （SCC） and 18 adenocarcinomas （ADC）, including only 8 ADC with Barrett esophageal columnar epithelium （metaplasia） and dysplasia adjacent to ADC. Paraffin-embedded normal squamous epithelium, Barrett esophageal columnar epithelium （metaplasia）, dysplasia and esophageal tumor tissues were dissected from the surrounding tissues under microscopic guidance. DNA was extracted using proteinase K digestion buffer, and DNA was diluted at 1：100, 1：1000, 1：5000, 1：10000 and 1：50000, respectively. Seven microsatellite markers （D2S123, D3S1616, D3S1300, D5S346, D17S787, D18S58 and BATRII loci） were used in this study. Un-dilution and dilution polymerase chain reactions （PCR） were performed, and microsatellite analysis was carried out.
RESULTS： No statistically significant difference was found in microsatellite instability （MSI） and loss of heterozygosity （LOH） of un-diluted DNA between SCC and ADC. The levels of MSI and LOH were high in the metaplasia-dysplasia-adenocarcinoma sequence of diluted DNA. The more the diluted DNA was, the higher the rates of MSI and LOH were at the above 7 loci, especially at D3S1616, D5S346, D2S123, D3S1300 and D18S58 loci.
CONCLUSION： The sequence of metaplasia-dysplasia-adenocarcinoma is associated with microsatellite alterations, including MSI and LOH. The MSI and LOH may be the early genetic events during esophageal carcinogenesis, and genetic alterations at the D3S1616, D5S346 and D3S123 loci may play a role in the progress of microsatellite alterations.     

Genetic changes in Slovenian patients with gastric adenocarcinoma evaluated in terms of microsatellite DNA

OBJECTIVES: Adenocarcinoma of the stomach is a relatively frequent malignant disease in Slovenia. We investigated the frequency of microsatellite instability (MSI) and loss of heterozygosity (LOH) in gastric carcinomas from the Slovenian population to determine their prognostic significance. METHODS: We evaluated MSI of mismatch repair associated loci and LOH on loci associated with following tumour suppressors: APC, nm23, Rb and p53. Results of the multiplex-PCR amplifications were correlated with clinicopathological factors for 73 patients. RESULTS: LOH was found in 52% of informative samples (20.5% LOH-H; 31.5% LOH-L). We found correlation of MSI with low-frequency LOH (LOH-L) in 11% of cases and with high-frequency LOH (LOH-H) tumours in 4% of cases. LOH-H and high-frequency MSI (MSI-H) were not associated. LOH was found in APC 36%, p53 33%, Rb 24% and nm23 33% of informative samples, whereas MSI was found in 30% of samples (12% MSI-H; 18% MSI-L). LOH-H status was associated with ulceration (P=0.029). LOH-N status was associated with diagnosis at higher TNM status (0.074) and infiltrative growth (P=0.006). Interestingly, in 6% of samples we found MSI on LOH loci as well. MSI-H was associated with higher age at diagnosis (r=0.24; P=0.04), antral location (r=0.252; P=0.04), intestinal type (P=0.044), expansive growth (P=0.001), tubular type (0.014), better differentiation (P=0.01), less nodal involvement (0.006) and better survival (P=0.022). The poorest prognosis was found in patients with both low-frequency MSI (MSI-L) and low-frequency LOH (LOH-L) tumours. CONCLUSION: The experimental design presented in the study may be of potential value for clinicians: at least five relevant markers for both MSI and LOH analysis may be needed to evaluate a gastric cancer (GC) patient's clinical status.     

Clinicopathological significance of loss of heterozygosity and microsatellite instability in hepatocellular carcinoma in China

AIM: To determine the features of microsatellite alterations and their association with clinicopathological characteristics of hepatocellular carcinoma (HCC). METHODS: Loss of heterozygosity (LOH) and microsatellite instability (MSI) of 55 microsatellite loci were detected with PCR-based microsatellite polymorphism analyses in tumors and corresponding noncancerous liver tissues of 56 surgically resected HCCs using the MegaBACE 500 automatic DNA analysis system. RESULTS: LOH was found in 44 of 56 HCCs (78.6%) at one or several loci. Frequencies of LOH on 1p, 4q, 8p, 16q, and 17p were 69.6% (39/56), 71.4% (40/56), 66.1% (37/56), 66.1% (37/56), and 64.3% (36/56), respectively. MSI was found in 18 of 56 HCCs (32.1%) at one or several loci. Ten of fifty-six (17.9%) HCCs had MSI-H. Serum HBV infection, alpha-fetoprotein concentration, tumor size, cirrhosis, histological grade, tumor capsule, as well as tumor intrahepatic metastasis, might be correlated with LOH on certain chromosome regions. CONCLUSION: Frequent microsatellite alterations exist in HCC. LOH, which represents a tumor suppressor gene pathway, plays a more important role in hepatocarcin-ogenesis. MSI, which represents a mismatch repair gene pathway, is a rare event during liver carcinogenesis. Furthermore, LOH on certain chromosome regions may be correlated with clinicopathological characteristics in HCC.     

肝细胞癌4号染色体微卫星变异的研究

目的 探讨肝细胞癌(HCC)微卫星变异的特点及其与临床病理的相关性。方法 应用聚合酶链反应-简单重复序列多态性方法,对56例患者HCC中4号染色体上10个微卫星的杂合性缺失(LOH)、微卫星不稳定性(MSI)和等位基因失衡(AI)3种变异特征进行检测。结果 56例HCC中,LOH的频率为71.4％(40/56),D4S426的LOH率最高为61.0％,其次为D4S1534(53.7％)。D4S406基因座,血清乙型肝炎表面抗原阳性患者的LOH频率高于阴性者[76.9％(20/26)与12.5％(2/16),x~2=13.999,P<0.01];在D4S426、D4S1615和D4S1652基因座,EdmondsonⅢ、Ⅳ级的LOH明显高于Edmondson Ⅰ、Ⅱ级[76.7％(23/30)与18.2％(2/11),x~2=9.242、P<0.01;53.8％(14/26)与16.7％(2/12),P<0.05;60.7％(17/28)与18.2％(2/11),P<0.051;D4S2921基因座,肝内转移者的LOH显著高于无肝内转移者[63.6％(21/33)与18.2％(2/11),x~2=5.132,P<0.05]。MSI的频率为8.9％(5/56);AI的频率为26.8％(15/56)。结论 HCC 4号染色体微卫星变异形式以LOH为主,提示LOH路径在HCC的发生和发展过程中起主要作用,MSI路径的作用次之。     

燃煤型砷中毒患者皮损PTCH基因D9S287和D9S180位点微卫星不稳定性的观察

目的观察燃煤型砷中毒患者皮损组织中PTCH基因微卫星DNA不稳定性及杂合性丢失与临床病理、临床分度之间的关系。方法选取D9S287、D9S180两个微卫星多态性标记,采用PCR扩增-变性聚丙烯酰胺凝胶电泳-银染法检测不同病理类型的燃煤型砷中毒患者的微卫星的改变。结果34例患者皮损组织PTCH基因微卫星不稳定性的发生率为29.41%(10/34),杂合性丢失的发生率为14.7%(5/34),微卫星的改变与病理分型相关(P<0.01),与临床分度无关(P>0.05)。结论PTCH基因微卫星不稳定性和杂合性丢失可能在砷中毒患者皮损癌变的发生发展中起重要作用。     

Genetic instability in myelodysplastic syndrome: detection of microsatellite instability and loss of heterozygosity in bone marrow samples with karyotype alterations

Using a polymerase chain reaction (PCR)-based approach, we examined the prevalence of loss of heterozygosity (LOH) and microsatellite instability (MSI) in relation to chromosomal imbalances in myelodysplastic syndrome (MDS). Two of 26 patients displayed MSI (8%), one of them at five loci. LOH was detected in six out of 26 cases (23%), predominantly involving markers IRF1 [5q31] and WT1 [11p]. Two patients displayed a corresponding chromosomal deletion by conventional cytogenetics. Supporting the mutator phenotype hypothesis, a significant coincidence of LOH, MSI and chromosome abnormalities was observed (P < 0.025). Moreover, our data suggest that LOH represents an initial rather than a secondary genetic event in MDS, promoting genetic instability in a subset of patients.     

肝癌患者血浆循环DNA杂合性缺失的检测及其临床意义

目的检测HCC患者血浆循环DNA杂合性缺失（LOH），并探讨将其作为有关临床预测标记的可能性。方法选择位于染色体8p上3个具有高度多态性的微卫星标记D8S277、D8S298和D8S1771，对62份HCC患者血浆循环DNA进行LOH检测，并进一步探讨LOH与患者HBsAg表达、是否肝硬化、血清AFP水平、肿瘤大小及细胞分化程度和有无肝内转移等临床病理特征之间的关系。结果在62份HCC患者血浆循环DNA标本中，36份（58．1％）在1个或多个位点发生LOH，D8S277、D8S298和D8S1771位点杂合度分别为74．2％（46／62）、75．8％（47／62）和69．4％（43／62），LOH频率分别为32．6％（15／46）、44．7％（21／47）和46．5％（20／43）。D8S298位点有肝内转移患者血浆循环DNA标本的LOH频率（62．5％）明显高于无肝内转移者（26．1％），差异有统计学意义（P〈0．05）；其他临床病理特征与3个位点上的LOH频率无明显相关。结论血浆循环DNA中D8S298位点LOH有可能成为HCC术后转移复发及预后的一个潜在的预测标记。     

Detection of loss of heterozygosity by high-resolution fluorescent system in non-small cell lung cancer: association of loss of heterozygosity with smoking and tumor progression

BACKGROUND: We recently developed a novel system for detecting microsatellite alteration, which is an important process in carcinogenesis. In patients with non-small cell lung cancer (NSCLC), loss of heterozygosity (LOH) is frequently observed and causes functional disorders of tumor suppressor genes. PATIENTS AND METHODS: In a consecutive series of 51 patients with NSCLC who had undergone a surgical resection, microsatellite instability (MSI) and LOH in tumors were analyzed by polymerase chain reaction using five fluorescence-labeled dinucleotide markers (D2S123, D5S107, D10S197, D11SS904, and D13S175) and an autosequencer. RESULTS: MSI was detected in only one patient (2.0%) with only one marker. LOH was detected in at least one chromosomal region that was tested in 39 patients (76%). The mean (+/- SD) number of LOHs detected by each marker was 1.74 +/- 1.40, with 1 LOH detected in 10 patients, 2 LOHs detected in 15 patients, 10 LOHs detected in 3 patients, 1 LOH detected in 4 patients, and 3 LOHs detected in 5 patients. The number of LOHs detected in each patient was significantly associated with the pack-year index (rho = 0.501; p = 0.0004), although there was no relationship with having a history of multiple cancers and familial cancer. Patients with stage IA disease showed a significantly lower number of LOHs than did patients with other stages of disease (1.15 vs 2.38, respectively; p = 0.0013). CONCLUSION: LOH is very common in patients with NSCLC, and the number of LOHs increases with increases in smoking, suggesting the presence of an important event in lung carcinogenesis.     

Microsatellite analysis of induced sputum DNA in patients with lung cancer in heavy smokers and in healthy subjects

Abnormality in the fragile histidine triade (FHIT), a candidate tumor suppressor gene located in chromosome region 3 (3p14.2), has been frequently found in multiple tumor types, including lung cancer. In this study, the authors assessed the consistency of DNA microsatellite analysis of induced sputum (IS), as compared to that of blood and plasma. They also evaluated the loss of heterozigosity (LOH) and microsatellite instability (MSI) in 3 different loci, D3S1300, D3S1313, and D3S1234, all internal to the FHIT gene, in IS, blood, and plasma from patients with lung cancer, smokers, and healthy subjects. Eighteen patients with lung cancer (3 females, age mean +/- SD: 63 +/- 7 years), 39 smokers (23 females, age mean +/- SD: 57 +/- 6 years and cigarette pack-years mean +/- SD: 34 +/- 12), and 22 healthy nonsmoking subjects (13 females, age mean +/- SD: 63 +/- 5 years) were studied. DNA was extracted from blood, plasma, and IS, by means of a standard method. Analysis of LOH and MSI were performed using a fluorescent polymerase chain reaction (PCR)-based approach, followed by capillary electrophoresis. The ratios between the peak heights (phs), expressed as random fluorescence units, from plasma/blood (p/b) and induced sputum/blood (is/b) in all three loci were considered. The biases (agreement limits) between the mean ph ratio from p/b and is/b of D3S1300, D3S1313, and D3S1234 were respectively 0.07 (- 0.39 to 0.53), 0.016 (- 0.32 to 0.35), - 0.10 (- 0.51 to 0.30) in the patients; - 0.04 (- 0.52 to 0.43), - 0.06 (- 0.31 to 0.18), - 0.08 (- 0.48 to 0.30) in smokers; and - 0.11 (- 0.40 to 0.17), - 0.05 (- 0.53 to 0.43), - 0.09 (- 0.51 to 0.33) in healthy subjects. LOH and MSI in at least one locus were observed in 55% of patients, in 18% of smokers, and in 4.5% of healthy subjects (P < 0.001). These results showed that IS DNA provided data that were consistent with those from blood and plasma. These findings highlight new prospects for early tumor detection by a noninvasive technique based on the analysis of genetic alterations in induced sputum.     

Loss of heterozygosity and microsatellite instabilities of fragile histidine triad gene in gastric carcinoma

AIM: To detect the loss of heterozygosity (LOH) and microsatellite instabilities (MSI) of fragile histidine triad (FHIT) gene in gastric carcinoma and to study their association with the clinical pathological characteristics of gastric carcinoma. METHODS: LOH and MSI of FHIT gene were detected at four microsaterllite loci D3SI3H, D3S4I03, D3SI48I and D3S1234 using PCR in matched normal and cancerous tissues from 50 patients with primary gastric cancer. RESULTS: The average frequency of LOH and MSI of FHIT gene in gastric cancer was 32.4% and 26.4% respectively. LOH and MSI of FHIT gene in gastric cancer had no association with histological, Borrmann, and Lauren's classification. LOH of FHIT gene in gastric cancer was related to invasive depth. The frequency of FHIT LOH in gastric cancer with serosa-penetration was obviously higher than that in gastric cancer without serosa-penetration (73.5% vs 37.5%, P < 0.05). MSI of FHIT gene in gastric cancer was associated with the lymph node metastasis. The frequency of MSI in gastric cancer without lymph node metastasis was significantly higher than that in gastric cancer with lymph node metastasis (66.7% vs 34.3%, P < 0.05). CONCLUSION: LOH of FHIT gene is correlated with invasive depth of gastric carcinoma. MSI of FHIT gene is correlated with lymph node metastases. LOH and MSI of FHIT gene play an important role in carcinogenesis of gastric cancer.     

Microsatellite instability in gallbladder carcinoma: two independent genetic pathways of gallbladder carcinogenesis

Although the genetic basis for gallbladder carcinogenesis has not been clarified, considerable evidence has shown that genetic alterations play an important role in the development and progression of human cancers. In this study, we analyzed 30 gallbladder carcinomas to investigate the role of genetic alterations in their tumorigenesis, and to study correlations with their clinicopathological features. Tissue samples were obtained from 30 patients with gallbladder carcinoma (11 men and 19 women; mean age, 62 years; age range, 38–80 years). Genomic DNAs were extracted from fresh tumor tissue. We examined loss of heterozygosity (LOH) in the p53, APC, DCC, RB, and NM23-H1 gene regions by polymerase chain reaction (PCR)-LOH assay using an automated fluorescent DNA sequencer employing four microsatellite markers (p53, APC, DCC, NM23-H1). Five additional microsatellite markers were used for the determination of microsatellite instability (MSI). LOH was found at p53 in 9 of 15 informative cases (60%), at DCC in 10 of 22 (45%), at APC in 5 of 15 (33%), at RB in 1 of 8 (13%), and at NM23-H1 in 1 of 15 (7%). MSI was observed in 5 of 30 cases (17%) in at least one chromosomal loci of these nine microsatellite markers. None of the patients with MSI-positive tumors showed lymph node metastasis, and there was an inverse correlation between MSI and the presence of LOH in gallbladder carcinoma. These results suggest that there are two independent genetic pathways in gallbladder carcinogenesis; that is, an MSI pathway and an LOH pathway. Received: December 24, 1999 / Accepted: May 26, 2000     

Loss of heterozygosity： An independent prognostic factor of colorectal cancer

AIM: Colorectal cancers result from the accumulation of several distinct genetic alterations. This study was to investigate the frequency and prognostic value of loss of heterozygosity (LOH) and microsatellite instability (MSI) at 14 genetic loci located near or within regions containing important genes implicated in colorectal tumorigenesis. METHODS: We studied colorectal cancers with corresponding normal mucosae in 207 patients (139 males and 68 females, mean age at the time of tumor resection 66.2±12.4 years, range 22-88 years). There were 37 right-sided colonic tumors, 85 left-sided colonic tumors and 85 rectal tumors. The distribution of tumor staging was stage Ⅰ in 25, stage Ⅱ in 73, stage Ⅲ in 68, and stage Ⅳ in 41. We analyzed the LOH and MSI of HPC1, hMSH2, hMLH1, APC, MET, P53, NH23-H1, DCC, BAT25, BAT26, D17S250, MYCL1 and D8S254 with fluorescent polymerase chain reaction and denatured gel electrophoresis. High-frequency LOH was determined to be greater than three, or more than 50% of the informative marker with LOH. High-frequency MSI (MSI-H) was determined as more than four markers with instability (>30%). Correlations of LOH and MSI with clinical outcomes and pathological features were analyzed and compared. RESULTS: The occurrence of MSI-H was 7.25%, located predominantly in the right colons (7/15) and had a higher frequency of poor differentiation (6/15) and mucin production (7/15). LOH in at least one genetic locus occurred in 78.7% of the tumors and was significantly associated with disease progression. Of the 166 potentially cured patients, 45 developed tumor recurrence within 36 mo of follow-up. Clinicopathological factors affecting 3-year disease-free survival (DFS) were TNM staging, grade of differentiation, preoperative CEA level, and high LOH status. Patients with high LOH tumors had a significantly lower DFS (50%) compared with patients with low LOH tumors (84%). Of the patients developing subsequent tumor recurrence, the number and percentage of LOH were 2.97 and 46.8% respectively, similar to the stage IV disease patients. TNM staging had the most significant impact on DFS, followed by high LOH status. CONCLUSION: Clinical manifestations of LOH and MSI are different in colorectal cancer patients. High-frequency LOH is associated with high metastatic potential of colorectal cancers.     

胃癌微卫星不稳定性和抑癌基因杂合缺失

目的研究微卫星不稳和抑癌基因缺失在胃癌发生中的作用．方法采用ＰＣＲ为基础的方法,检测了５３例胃癌中６个微卫星标记突变及ＡＰＣ／ＭＣＣ和ＤＣＣ基因杂合缺失（ＬＯＨ）．结果胃癌微卫星不稳的检出率为３２１％（１７／５３）．７例（１３２％）为微卫星高频率不稳（３个以上微卫星标志）,１０例（１８９％）为微卫星低频率不稳（１或２个微卫星标记）．肠型胃癌微卫星高频率不稳的发生率（２５０％）显著高于弥漫型胃癌（３４％）（Ｐ＜００５）．高频率不稳组未发现有ＡＰＣ,ＭＣＣ和ＤＣＣ基因ＬＯＨ,微卫星高频率不稳与ＡＰＣ／ＭＣＣ和ＤＣＣ基因ＬＯＨ呈负相关．结论微卫星不稳在部分胃癌,特别是肠型胃癌早期发生中起重要作用,高频率不稳胃癌与遗传性非息肉大肠癌有共同的特点．与此相反,低频率不稳和无不稳胃癌可能通过ＬＯＨ病理途径发生     

Are there tumor suppressor genes on chromosome 4p in sporadic colorectal carcinoma？

AIM： To study the candidate tumor suppressor genes （TSG） on chromosome 4p by detecting the high frequency of loss of heterozygosity （LOH） in sporadic colorectal carcinoma in Chinese patients. METHODS： Seven fluorescent labeled polymorphic microsatellite markers were analyzed in 83 cases of colorectal carcinoma and matched normal tissue DNA by PCR. PCR products were eletrophoresed on an ABI 377 DNA sequencer. Genescan 3.7 and Genotype 3.7 software were used for LOH scanning and analysis. The same procedure was performed by the other six microsatellite markers spanning D4S3013 locus to make further detailed deletion mapping. Comparison between LOH frequency and clinicopathological factors was performed by 2 test. RESULTS： Data were collected from all informative loci. The average LOH frequency on 4p was 24.25%, and 42.3% and 35.62% on D4S405 and D4S3013 locus, respectively. Adjacent markers of D4S3013 displayed a low LOH frequency （〈 30%） by detailed deletion mapping. Significant opposite difference was observed between LOH frequency and tumor diameter on D4S412 and D4S1546 locus （0% vs 16.67%, P = 0.041; 54.55% vs 11.11%, P = 0.034, respectively）. On D4S403 locus, LOH was significantly associated with tumor gross pattern （11.11%, 0, 33.33%, P = 0.030）. No relationship was detected on other loci compared with clinicopathologial features. CONCLUSION： By deletion mapping, two obvious high frequency LOH regions spanning D4S3013 （4p15.2） and D4S405 （4p14） locus are detected. Candidate TSG, which is involved in carcinogenesis and progression of sporadic colorectal carcinoma on chromosome 4p, may be located between D4S3017 and D4S2933 （about 1.7 cm）.     

慢性白血病微卫星不稳定性和杂合性缺失的研究

应用PCR扩增技术检测17例慢性白血病患者不同染色体上8个微卫星位点的微卫星不稳定性（MSI）和杂合性缺失（LOH）,同时检测11例健康志愿者的MSI和LOH,以作对照。结果发现,健康对照组所选微卫星位点均未发生MSI或LOH;9例处于慢性白血病加速期的人中有7例发生至少一个位点的MSI,8例慢性期患者中仅1例发生一个位点的MSI,慢性白血病加速期的MSI发生率明显高于其慢性期（P＜0.05>,提示微卫星的遗传不稳定性可能与慢性白血病的病情进展有关。17例慢性白血病中只有1例在急变后出现LOH,提示所选位点的LOH可能不是慢性白血病的多发事件。     

Differences in microsatellite DNA level between asthma and chronic obstructive pulmonary disease.

Previous studies have shown that microsatellite (MS) DNA instability (MSI) is detectable in sputum cells in chronic obstructive pulmonary disease (COPD) and asthma. The aim of the present study was to investigate whether asthma and COPD could be distinguished at the MS DNA level. DNA was extracted from sputum cells and white blood cells from 63 COPD patients, 60 non-COPD smokers, 36 asthmatics and 30 healthy nonsmokers. Ten MS markers located on chromosomes 2p, 5q, 6p, 10q, 13q, 14q and 17q were analysed. No MSI was detected in non-COPD smokers or healthy nonsmokers. A significantly higher proportion of COPD patients exhibited MSI (49.2%) compared to asthmatics (22.2%). MSI was detected even in the mild stages of COPD (33.3%) and asthma (22.2%). No relationship was found between MSI and COPD severity. The most frequently affected marker was D14S588 (17.5% in COPD and 2.7% in asthma). The markers D6S344, G29802 and D13S71 showed alterations only in COPD, and G29802 was associated with a significantly decreased forced expiratory volume in one second FEV1 (% predicted), whereas MSI in D6S344 was associated with a significantly higher FEV1 (% pred). The frequency of microsatellite instability was higher in chronic obstructive pulmonary disease than in asthma, and microsatellite instability in three workers showed chronic obstructive pulmonary disease specificity. However, further studies are needed to verify the differences between chronic obstructive pulmonary disease and asthma at the microsatellite level.     

Microsatellite instability in Ewing tumor is not associated with loss of mismatch repair protein expression

Only few clinical factors predict the prognosis of patients with Ewing tumors. Unfavorable outcome is associated with primary metastatic disease, age > 15 years, tumor volume above 200 ml, and the histological response to chemotherapy. The aim of this study was to elucidate the prevalence and clinical impact of microsatellite instability (MSI) together with the relation between MSI and mismatch repair protein expression in Ewing tumors. DNA from 61 primary Ewing tumors and 11 Ewing tumor cell lines was extracted and microsatellite analysis for the detection of instability or loss of heterozygosity was performed for the five markers of the Bethesda panel BAT25, BAT26, D5S346, D2S123, and D17S250, which represents the established marker panel for the analysis of hereditary non-polyposis colorectal carcinoma (HNPCC) patients. In addition, single nucleotide repeat regions of the two tumor genes BAX and transforming growth factor receptor II (TGFBR2) were also included. All of the 61 samples were suitable for LOH analysis and 55 for the determination of MSI-status. LOH of these microsatellite markers was detected in 9 of the 61 patients (14.8%). Over all, genetic instability, i.e. MSI and/or LOH, was detected in 17 tumors (27.9%). One out of the 11 tumor cell lines (STA ET1) was characterized by instability of all the five Bethesda markers, while from primary tumor samples, only one showed MSI in more than one microsatellite marker (D5S346 and D17S250, MSI-high). Eight of the fifty-five patients (14.5%) showed instability of one microsatellite locus (MSI-low). No instability was detected in BAT26, D2S123, BAX and TGFBR2. There was no significant correlation between MSI and loss of expression of mismatch repair proteins MLH1, MSH2, or MSH6. The impairment of the p53 signaling pathway (expression of TP53 and/or MDM2 by immunohistochemistry) was significantly associated with reduced overall survival (15 of 49 patients (30.6%), P = 0.0410, log-rank test). We conclude that MSI is not prevalent in Ewing tumor and that the nature of instability differs from the form observed in colorectal carcinoma, the model tumor of MSI. This is documented by the different pattern of MSI (no BAT26 instability) in Ewing tumors and the lack of a strict correlation between MSI-high and loss of expression of MSH2, MSH6 and MLH1. IA and KLS contributed equally to this study.