Microsatellite instability in gastric cancer with varied structure

BACKGROUND: The intratumoral histological heterogeneity of cancer has been investigated by many pathologists. Although microsatellite alteration has been reported in gastric cancer, the significance of genomic instability in these histologically heterogeneous cases has not been elucidated. METHODS: Microsatellite alteration detected by polymerase chain reaction (PCR) in 13 primary advanced gastric cancers with varied structure was examined at 8 microsatellite loci. RESULTS: We were able to detect a greater prevalence of replication errors (RER) (6/13, 46.2%) at the primary site of gastric cancer than previously reported and loss of heterozygosity (LOH) at 17p (4/13, 30.8%) was demonstrated at the primary sites. All the same time, we also examined metastatic tumors in the regional lymph nodes in 12 of these cases. The frequency of RER (8/12, 66.7%) in metastatic lesions was higher than that in primary tumors. Detectability of RER was more frequent in the poorly differentiated portions than the well-differentiated portions of lymph node metastases. CONCLUSIONS: These findings suggest that gastric cancer with varied structure acquired frequent microsatellite instability during progression and metastasis, and we reasoned that the mutator phenotype detected by microsatellite alterations may represent heterogeneous tumor clones in gastric cancer and lymph node metastases.     

Microsatellite analysis of plasma DNA from patients with clear cell renal carcinoma

Deletions of DNA sequences on chromosome 3p [loss of heterozygosity (LOH)] are characteristic of clear cell renal carcinoma, which accounts for about 80% of all renal malignancies. Comparing tumor DNA to DNA from normal cells, LOH analysis of microsatellite sequences has aided in molecular diagnosis of renal carcinoma. Because clinically useful tumor markers do not exist for this cancer entity, the aim of the present study was to detect chromosome 3p microsatellite alterations (LOH and microsatellite instability) in plasma DNA from patients with clear cell renal carcinoma. Four chromosome 3p microsatellites (D3S1307, D3S1560, D3S1289, and D3S1300) were amplified by fluorescent PCR using DNA isolated from normal blood cells and plasma of 40 patients. Corresponding tumor DNA was available from 21 patients. Analyzing PCR products on an automated DNA sequencer, we found LOH in at least one locus in 25 plasma samples (63%), and 14 plasma samples (35%) exhibited LOH at more than one locus. Microsatellite instability of plasma DNA was detectable in one patient (3%). No significant association of advanced (>T2N0M0) tumor stages with LOH in plasma DNA could be demonstrated. If present, modifications of plasma DNA and tumor DNA were identical. No alterations of plasma DNA were found in healthy controls. Analysis of plasma DNA from patients with clear cell renal carcinoma reveals tumor-specific microsatellite alterations and may therefore have diagnostic potential as a molecular tumor marker.     

In memoriam: Widukind Lenz, 1919-1995

To determine whether microsatellite instability is involved in the development of transitional cell carcinoma (TCC) of the urinary tract, a microsatellite instability assay was carried out using PCR with 9 microsatellite loci. Thirty-eight TCC samples (30 patients with bladder cancer, 5 with renal pelvic tumors and 3 with ureteral tumors) and 1 lymph node with metastasis were examined. Microsatellite instability was found in 8 of 38 tumors examined, and 3 showed alterations in more than 2 microsatellite loci. All 8 tumors were beyond grade 2 and stage pT2 advanced tumors. Stages pT1-2 and pT3-4 patients differed significantly. Microsatellite instability was greater in smokers than non-smokers, but the differences were not significant. Microsatellite instability in TCC of the urinary tract is rare in superficial tumors but more common in invasive tumors. Microsatellite alterations would thus appear to occur, and possibly be importantly involved, in the tumorigenesis of urinary tract TCC.     

Loss of heterozygosity on chromosome arm 3p in nasopharyngeal carcinoma

We have examined 17 primary undifferentiated nasopharyngeal carcinoma biopsies for allelic loss on 3p, comparing the findings in tumors with those in normal lymphocyte DNA from the same patients. Ten polymorphic microsatellite markers were used between 3p13 and 3p26. Allelic loss was observed in 12 samples (70%). Two loci were most frequently affected: D3S1067 (3p21.1-14.3) in 60% and D3S1217 (3p14.2-14.1) in 58%. One tumor seemed to have a homozygous deletion at 3p26, detected by the D3S1297 marker. Analysis of the clinical data showed that an increased number of aberrations in 3p was correlated with more advanced tumor stages.     

Multifocal renal cell carcinoma: evidence for a common clonal origin

The reported incidence of satellite tumor lesions in kidneys resected by radical nephrectomy for renal cell carcinoma (RCC) is 7-25%; however, genetic analyses of satellite tumors in comparison with those of main tumor lesions have not been performed well. In the present study, we investigated the incidence of loss of heterozygosity (LOH) at chromosome arms 3p, 6q, 8p, 9p, 9q, and 14q using 18 microsatellite markers in 10 nonpapillary RCCs of 50 mm or less in diameter and the accompanying satellite tumor lesions to evaluate the genetic alterations in main and satellite tumors. LOH was detected in 10, 3, 5, 3, 2, and 3 cases at chromosome arms 3p, 6q, 8p, 9p, 9q, and 14q, respectively. In addition, primary and satellite tumor lesions in 8 of 10 cases exhibited identical patterns of LOH on the 18 loci examined. In the remaining two cases, both main and satellite tumors demonstrated LOH on the common seven and three loci, respectively, whereas for another locus, LOH was observed only in the satellite tumor lesions. The similarity of LOH patterns detected in main and satellite tumor lesions indicates that the presence of satellite tumors might be the result of intrarenal metastasis from the main tumor lesion. These findings strongly suggest that even in case of small nonpapillary RCC, nephron-sparing surgery might carry the risk of failing to prevent postoperative local recurrence due to the incomplete resection of unrecognized satellite tumors with genetic alterations similar to those of the main tumor.     

Respiratory symptoms and spirometry in experienced coal miners: effects of both distant and recent coal mine dust exposures

Loss of heterozygosity (LOH) on 3p is frequent in human renal cell carcinomas, lung cancers, and breast cancers. To define the region(s) on 3p that harbor presumptive tumor suppressor gene(s) for breast cancer, we examined 196 primary breast tumors for their patterns of LOH at 22 microsatellite marker loci distributed along this chromosome arm. Allelic loss at one or more loci was observed in 101 (52%) of these tumors. Detailed deletion mapping identified two distinct commonly deleted regions; one was localized to a 2-cM interval flanked by D3S1547 and D3S1295 at 3p14.3-21.1, and the other to a 5-cM interval flanked by D3S1286 and D3S1585 at 3p24.3-25.1. The FHIT gene lies in the vicinity of the proximal commonly deleted region. Attempts to correlate LOH on 3p to clinicopathological parameters detected an association with the absence of the progesterone receptor (P = 0.0096). The results suggest that inactivation of unidentified tumor suppressor genes on 3p plays a role in the mechanism whereby hormone dependency is lost in the course of breast carcinogenesis.     

Ethnic differences in interferon-alpha allele frequencies

We analyzed microsatellite instability (MSI) and loss of heterozygosity (LOH) at 17 microsatellite markers located on chromosomes 2p, 3p, 5q, 6q, 9p, 9q, 17p and 18q in 19 randomly selected keratoacantomas (KAS), in one cutaneous lesion that histologically could not unequivocally be differentiated from squamous cell carcinoma, and in one patient with multiple KAs of longstanding duration. The goals of our study were to determine whether, in a similar manner to some visceral carcinomas, genomic instability could be detected in KAs and to clarify whether molecular analysis might be useful to further characterize KA. MSI was observed in 2 of 21 cases (9.5%) at 5 of 17 loci examined. In one patient with a solitary KA, the presence of MSI and a family history of visceral malignant tumours suggested that the patient might have belonged to a family with Muir-Torre syndrome. In one other MSI+ KA, a definite differential diagnosis in relation to squamous cell carcinoma could not be established. In addition, one sample displayed LOH at 2 of 17 loci analysed whereas in the patient with multiple KAs, LOH at one locus was the only alteration found. In conclusion, the low frequency of MSI and LOH detected in our study suggests that these genetic events are uncommon in KA unless it is associated with a familial disease (e.g. Muir-Torre syndrome) or it has more aggressive histological features.     

Human bladder cancers and normal bladder mucosa present the same hot spot of heterozygous chromosome-9 deletion

Loss of heterozygosity (LOH) on chromosome 9 is the most frequent genetic alteration in bladder cancer identified to date, suggesting the presence of key gene(s) for this pathology. In this study, we examined 44 bladder tumors and 21 normal bladder samples for LOH on both arms of chromosome 9. Sixteen microsatellite markers, 12 on the short arm (encompassing 9p21-22) and 4 on the long arm (encompassing 9q33-34), were chosen for their highly frequent alterations in bladder cancer. LOH for at least one marker was identified in 42 tumor samples (95.5%), and 14 tumors (32%) displayed LOH for all informative tested markers. Detailed analysis showed that 2 markers on chromosome 9p (D9S157 and D9S156) had the highest frequencies of allelic loss (about 70%), independent of tumor grade and stage. The same study was performed on the 21 normal bladder mucosa samples: 50% of informative cases presented a single specific LOH at the D9S156 locus. Normal samples showing LOH at this locus were therefore screened with 3 novel microsatellite markers in the 810-kb region incorporating D9S156. Using this marker, we found no further heterozygous loss in this region. This result allows different interpretations of the D9S156 loss in normal bladder mucosa, and suggests that D9S156 may be more an indicator of bladder epithelium impairment than a tumor-initiation marker. Similarly, this unexpected result calls in question the interpretation of LOH studies.     

Molecular analysis of childhood primitive neuroectodermal tumors defines markers associated with poor outcome

PURPOSE: The diagnostic and prognostic significance of well-defined molecular markers was investigated in childhood primitive neuroectodermal tumors (PNET). MATERIALS AND METHODS: Using microsatellite analysis, Southern blot analysis, and fluorescence in situ hybridization (FISH), 30 primary tumors and six CSF metastasis specimens were analyzed for loss of heterozygosity (LOH) of chromosomes 1q31, 6q, 9q22, 10q, 11, 16q22, and 17p13.1 and/or high-level amplification of the c-myc gene. Experimental data were compared with clinical stage and outcome. RESULTS: LOH of chromosome 17p13.1 was found most frequently (14 of 30 tumors, six of six CSF metastasis specimens); LOH of chromosomes 10q, 16q22, 11, 6, 9q22, and 1q31 was observed in 20.6%, 20%, 14.3%, 12%, 10%, and 0%, respectively. Eight of 32 tumors and CSF specimens showed amplification of c-myc. All tumors with amplification of c-myc were resistant to therapy and had a fatal outcome (mean survival time, 9.3 months). Tumors that displayed LOH of chromosome 17p were associated with metastatic disease. The prognosis of these tumors was worse only when associated with amplification of c-myc. Three of three patients with LOH of 9q22 relapsed. CONCLUSION: In our study, amplification of c-myc was a poor-prognosis marker in PNET. LOH of chromosome 17p was associated with metastatic disease. Molecular analysis of primary tumors using these markers may be useful for stratification of children with PNET in future prospective studies. The other aberrations investigated were not of significant prognostic value, but may provide an entry point for future large-scale molecular studies.     

Loss of heterozygosity at 7q31 is a frequent and early event in prostate cancer

It is widely accepted that an accumulation of genetic alterations plays an important role in the genesis of human cancers, but little is known about prostate cancer in this respect. Recent studies have identified regions on chromosome arms 8p, 10q, 16q, and 18q that are frequently deleted in human prostate cancer. We have previously described a loss of heterozygosity (LOH) at the Met locus on chromosome band 7q31 in a study of 20 localized prostate tumors. To determine whether a region on the 7q arm is important in the initiation and/or progression of prostate cancer, prostate tissue from 13 patients with confined prostate tumors, 17 with local extracapsular extension, and 13 with metastatic forms were analyzed for LOH, using a DNA probe for RFLP (pMetH) and 8 CA microsatellite repeats (7 on 7q21-q33 and 1 on 7p). Twenty (47%) of the 43 cases studied showed LOH at one or more 7q loci. The most frequently deleted region was chromosome 7q31.1-7q31.2, whereas the centromeric locus on 7q21 was generally conserved. The percentage of LOH was normally distributed around the D7S480 locus. Moreover, the rate of LOH in the 7q31 region was lower in metastatic tumors than in localized tumors. These results strongly suggest the presence of a tumor suppressor gene on the chromosome band 7q31 with an important role in the early stages of prostate cancer.     

Localization of chromosome 8p regions involved in early tumorigenesis of oral and laryngeal squamous carcinoma

We analysed 30 primary invasive oral and laryngeal squamous carcinomas (SC), with concurrent dysplastic lesions, for genetic alterations at 15 microsatellite loci on the short arm of chromosome 8. Overall, loss of heterozygosity (LOH) was observed, in at least one informative locus, in 27% of the dysplastic lesions and in 67% of the invasive carcinomas. The highest frequency of allele losses in dysplasia (20% and 17%), and invasive carcinoma (40% and 48%) were detected in the same D8S298 and LPL-tet loci located on chromosomes 8p21 and 8p22 respectively. The minimal region with LOH was limited to 4.6 megaBases (mBs) at 8p22 and 7.1 mBs at 8p21. In addition, allelic losses in both dysplastic and corresponding invasive specimens were noted at the same loci in some tumors suggesting their emergence from a common preneoplastic clone. Allele losses correlated significantly with male gender, oral and laryngeal sites and high proliferative index. The data suggest that inactivation of tumor suppressor gene(s), within these loci, may constitute an early event in the evolution of oral and laryngeal SC.     

Microsatellite instability analysis: a multicenter study for reliability and quality control

The molecular biology section of the Hereditary Non-Polyposis Colorectal Cancer study group-Germany, instituted a multicenter study to test the reliability and quality of microsatellite instability (MSI) analysis. Eight laboratories compared MSI analyses performed on 10 matched pairs of normal and tumor DNA from patients with colorectal carcinomas. A variety of techniques were applied to the detection of microsatellite changes: (a) silver and ethidium bromide staining of polyacrylamide gels; (b) radioactive labeling; and (c) automated fluorescence detection. The identification of highly unstable tumors and tumors without MSI was achieved in high concordance. However, the interpretation of the band patterns resulted in divergent classifications at several microsatellite marker loci for a large fraction of this tumor/normal panel. The data on more than 30 primers per case suggest that the enlargement of the microsatellite panel to more than 10 loci does not influence the results. In this study, cases with MSI in less than 10% of loci were classified as microsatellite stable, whereas MSI was diagnosed in cases with more than 40% of all markers unstable. We propose that a panel of five microsatellite loci consisting of repeats with different lengths should be analyzed in an initial analysis. When less than two marker loci display shifts in the microsatellite bands from tumor DNA, the panel should be enlarged to include an additional set of five marker loci. The number of marker loci analyzed as well as the number of unstable marker loci found should always be identified. These criteria should result in reports of MSI that are more comparable between studies.     

Leukocyte migration in acute colonic inflammatory bowel disease: comparison of histological assessment and Tc-99m-HMPAO labeled leukocyte scan

Loss of heterozygosity (LOH) on chromosome 11 is frequently altered in various epithelial cancers. The present study was designed to investigate LOH on chromosome 11 in microdissected samples of normal prostatic epithelium and invasive carcinoma from the same patients. For this purpose, DNA was extracted from the microdissected normal and tumor cells of 38 prostate cancers, amplified by polymerase chain reaction PCR and analyzed for LOH on chromosome 11 using 9 different polymorphic DNA markers (D11S1307, D11S989, D11S1313, D11S898, D11S940, D11S1818, D11S924, D11S1336 and D11S912). LOH on chromosome 11 was identified in 30 of 38 cases (78%) with at least one marker. Four distinct regions of loss detected were: 1) at 11p15, at loci between D11S1307 and D11S989; 2) at 11p12, on locus D11S131 (11p12); 3) at 11q22, on loci D11S898, D11S940 and D11S1818; and 4) at 11q23-24, on loci between D11S1336 and D11S912. We found 25% of the tumors with LOH at 11p15; 39% had LOH at 11p12; 66% had LOH at 11q22; and 47% had LOH at 11q23-24. These deletions at 11p15, 11p12, 11q22 and 11q23-24 loci were not related to the stage or grade of the tumor.     

Allelic loss of 16q23.2-24.2 is an independent marker of good prognosis in primary breast cancer

Allelic loss, detected as a loss of heterozygosity (LOH) on the long arm of chromosome 16, is an early and frequent event in breast cancer. Despite this, the clinical significance of LOH on 16q has been very poorly studied. In this study, corresponding blood and tumor samples from 199 clinically well-characterized primary breast cancer patients were analyzed for LOH with the highly polymorphic microsatellite marker D16S511, located at 16q23.2-24.2. 61% of 168 informative tumors showed LOH. Univariate and multivariate analysis found a highly significant association between LOH at 16q23.2-24.2 and freedom from distant metastases, disease-free survival, and overall survival, respectively. No association was found with other clinical parameters such as menopausal status, tumor size, lymph node status, histopathology, and lymph node capsule invasion. This makes allelic loss of 16q23.2-24.2 an independent marker of good prognosis for primary breast cancer.     

Clonal heterogeneity in human esophageal squamous cell carcinomas on DNA analysis

Cancers are thought to arise through multistep accumulation of somatic mutations in the progeny of a single cell. Multiple mutations may induce molecular intratumor heterogeneity. Therefore, we examined molecular clonal heterogeneity in esophageal squamous cell carcinomas. Twenty-four esophageal squamous cell carcinomas and associated lymph node metastases were examined for microsatellite alterations, and abnormalities of the p53 and transforming growth factor-beta type II receptor (TGF-beta RII) genes. There were eight cases (33%) showing different patterns of loss of heterozygosity in primary tumors and metastatic lymph nodes with microsatellite markers. On the other hand, the abnormalities of p53 were identical in all these cases. No mutation was detected in the simple repeated sequences of the TGF-beta RII gene. These results indicate that molecular clonal heterogeneity exists in esophageal squamous cell carcinomas. Therefore, care is necessary in preoperative genetic diagnosis using biopsy samples.     

Molecular damage in the bronchial epithelium of current and former smokers

BACKGROUND: Most lung cancers are attributed to smoking. These cancers have been associated with multiple genetic alterations and with the presence of preneoplastic bronchial lesions. In view of such associations, we evaluated the status of specific chromosomal loci in histologically normal and abnormal bronchial biopsy specimens from current and former smokers and specimens from nonsmokers. METHODS: Multiple biopsy specimens were obtained from 18 current smokers, 24 former smokers, and 21 nonsmokers. Polymerase chain reaction-based assays involving 15 polymorphic microsatellite DNA markers were used to examine eight chromosomal regions for genetic changes (loss of heterozygosity [LOH] and microsatellite alterations). RESULTS: LOH and microsatellite alterations were observed in biopsy specimens from both current and former smokers, but no statistically significant differences were observed between the two groups. Among individuals with a history of smoking, 86% demonstrated LOH in one or more biopsy specimens, and 24% showed LOH in all biopsy specimens. About half of the histologically normal specimens from smokers showed LOH, but the frequency of LOH and the severity of histologic change did not correspond until the carcinoma in situ stage. A subset of biopsy specimens from smokers that exhibited either normal or preneoplastic histology showed LOH at multiple chromosomal sites, a phenomenon frequently observed in carcinoma in situ and invasive cancer. LOH on chromosomes 3p and 9p was more frequent than LOH on chromosomes 5q, 17p (17p13; TP53 gene), and 13q (13q14; retinoblastoma gene). Microsatellite alterations were detected in 64% of the smokers. No genetic alterations were detected in nonsmokers. CONCLUSIONS: Genetic changes similar to those found in lung cancers can be detected in the nonmalignant bronchial epithelium of current and former smokers and may persist for many years after smoking cessation.     

Solvent polarity and pH effects on the spectroscopic properties of neutral red: application to lysosomal microenvironment probing in living cells

To investigate the molecular mechanism of gastric carcinogenesis, we analyzed genetic instability and p53 gene mutations in 40 primary gastric carcinomas. Tumor samples were from untreated patients with no family history suggestive of genetic predisposition to cancer. We screened six microsatellite loci by the polymerase chain reaction (PCR) method, and exons 5-8 of the p53 gene by the PCR-based denaturing gradient gel electrophoresis and sequencing techniques. Microsatellite instability was detected in 32.5% (13/40), and gene mutations in 40% (16/40), of the tumors analyzed. No statistically significant associations were found between genetic alterations and clinico-pathological variables (with the exception of diffusion of lymph node metastases, which was inversely associated with the presence of microsatellite alterations; P < 0.01). Interestingly, a negative association was found between genetic instability and p53 gene mutations: 11 out of 13 tumors showing instability proved to carry a nonmutated p53 gene versus 2/13 carrying a mutated gene (P = 0.03). These observations suggest that genetic instability and p53 gene mutations play a crucial role in the gastric carcinogenic process, but likely act through distinct pathways during cancer development. However, genetic instability is not in and of itself neoplastic. Therefore, we investigated whether insertion/deletion mutations of the polyadenine tract within the transforming growth factor-beta type II receptor gene (TGF-beta RII) were frequently present in gastric tumors with an RER+ (replication error) phenotype. We found RII mutations in 8/40 (20%) samples: mutations were present in 7/13 (54%) RER+ tumors versus 1/27 (4%) RER- cases (P < 0.001).     

Reduced exploratory activity of audiogenic seizures susceptible Wistar rats

To search for the existence of a tumour-suppressor gene (TSG) associated with oral squamous cell carcinoma (SCC), PCR analysis of microsatellite polymorphisms corresponding to 14 loci which map to chromosome 7q21.3-qter was performed to screen 35 patients with oral SCC for loss of heterozygosity (LOH). LOH was observed in at least one of the loci in 19 of 34 (55.9%) informative cases. Among the loci tested, frequent LOH was restricted at D7S522 on chromosome 7q31.1, which was measured within 1 cM. Furthermore, we detected microsatellite instability (MI) in 11 of 35 (31.4%) cases tested. Our observations indicate that alterations of chromosome 7q are associated with oral SCC tumorigenesis and that 7q31.1 might harbour at least one putative TSG.     

Difference between clinic and daytime blood pressure is not a measure of the white coat effect

We investigated the clinicopathological significance of dendritic cell infiltration (DCsI) in esophageal squamous cell carcinoma and in regional lymph nodes of 88 patients. The expression of mutated p53 protein and the degree of positive cancer cells of proliferating cell nuclear antigen (PCNA labeling index) in tumors were analyzed as biological markers. These factors were compared with the degree of DCsI in tumors and in lymph nodes. The number of dendritic cells (DCs) were counted and scored as per mm2 in each case. The degree of DCsI of tumors with expression of p53 (19/mm2, n=50) was significantly lower than that of DCsI in 38 tumors without expression of p53 (27/mm2, P=0.0411). However, no significant correlation was detected between the PCNA labeling index and the degree of DCsI in 88 primary tumors (P=0.1273). The degree of DCsI in 53 metastatic lymph nodes (30/mm2) was significantly lower than that of DCsI in 264 cancer-free regional lymph nodes (48/mm2, P=0. 02). Although the degree of DCsI in tumors was not an independent prognostic factor for the 78 surviving patients (P=0.2647), the 3-year survival rate of patients in stage III and IV who underwent curative operation and who had tumors with high DCsI (>9/mm2, n=16, 72%) was significantly higher than that of the 24 patients who had tumors with low DCsI (< or = 9/mm2, 21%, P=0.008). These findings indicate that DCs infiltrated in and around the esophageal cancer may play a defensive role of the hosts against the tumors. This immune defense of the hosts might be an important prognostic factor for patients with advanced esophageal cancer. However, cancer cells which express a mutated p53 protein might regulate the function or activity of DCs.