Characterization of genetic alteration patterns in human esophageal squamous cell carcinoma using selected microsatellite markers spanning multiple loci

In order to identify representative genetic alterations in esophageal squamous cell carcinomas (ESCC) and useful markers for future early detection, 34 ESCC samples with neighboring normal epithelia and 30 esophageal biopsy samples from Linzhou, P.R. China, were studied. Of the 38 microsatellite markers selected, half were linked with tumor suppressors. More than 40% of the tumor samples showed loss of heterozygosity (LOH) in at least one of the eight markers, D3S1067 and D3S1561 (both linked to hMLH1 locus), FABP2, D4S1613, D9S171 (p14ARF, p15INK4b, p16INK4a loci), Rb1 (intron), p53-2 (intron), and NM23-H1. Most of the 38 microsatellite markers did not display microsatellite instability (MSI) in more than 30% of the tumor samples, except D9S942 (p14ARF, p15INK4b, p16INK4a loci) and Bat26, which showed frequency at 32 and 41%, respectively. Of all the ESCC samples examined, 20 samples exhibited LOH in 25% or more of the informative markers. Three samples displayed MSI in more than 30% of the markers, indicating that MSI might be an important event in these subset ESCC cases. Statistically significant correlations were found between LOH of the hMLH1 locus and the general LOH status of the sample, and between the LOH of the hMLH1 locus and p53 mutations. In addition, correlation was found between MSI in D3S1067/D3S1561 and the general MSI status in the samples. However, MSI in the introns of hMLH1 and hMSH2 were not correlated with the general MSI status of the tumors. LOH analysis was also performed in 30 esophageal biopsy samples containing precancerous lesions with matching blood samples using nine microsatellite markers selected from the above studies. LOH frequence ranged from 0 to 33% in informative cases, mostly in the 9p21 and p53 gene regions, suggesting these regions are possible targets of genomic instability in early stage ESCC carcinogenesis. The results demonstrate the degree of genetic alterations at different loci of the chromosomes. Some of the microsatellite markers may be useful for the early detection of ESCC.     

Genetic progression and divergence in superficial esophageal squamous cell carcinoma by loss of heterozygosity analysis

Esophageal squamous cell carcinoma (SCC) is one of the most common fatal carcinomas worldwide and has some of the most malignant characteristics among gastrointestinal tumors. Although a high frequency of loss of heterozygosity (LOH) for various genes has been observed in esophageal SCCs, these findings do not provide any information regarding the genetic pathways that may underlie the development and progression of this type of tumor. To clarify the temporal and topographic pathways in the genetic evolution of esophageal SCC, we microdissected multiple foci from superficial mucosal invasive foci of tumors. We then carried out LOH analyses of the microdissected neoplastic foci. Sixteen superficial esophageal SCCs were examined. Three to six carcinoma foci from each superficial esophageal SCC were individually microdissected. We used 12 oligonucleotide primer pairs specific for the microsatellite markers for which frequent LOH in esophageal SCC has been reported. All tumors exhibited LOH of at least three microsatellite loci. A frequent homogeneous LOH pattern was detected for TP53 (60%), D16S518 (43%) and D3S1234 (29%), suggesting that the loss of these alleles is an early event in the development of esophageal SCC. A heterogeneous LOH pattern was detected for D13S325 (87%), D10S559 (73%), D3S1568 (58%), D3S1234 (57%) and D3S1621 (56%), suggesting that the loss of these alleles is a late event in the development of esophageal SCC. All tumors showed the LOH pattern of single clonal neoplasms with genetic progression and divergence. In conclusion, by extensive sampling of SCC lesions with microdissection and LOH analysis of multiple chromosomal loci, we successfully demonstrated dynamic and successive accumulation of genetic alterations in early SCC.     

p53 Gene mutation and genetic instability in superficial multifocal esophageal squamous cell carcinoma

Tumor multicentricity is occasionally observed in esophageal squamous cell carcinoma (SCC). We studied five surgically resected superficial multifocal esophageal SCCs for p53 gene mutation and genetic instability, using DNA extracted from microdissected areas. A total of 38 target areas (TAs) were analyzed in SCC, dysplasia, basal cell hyperplasia (BCH) and normal squamous epithelium. Analysis of the replication error (RER) at 10 microsatellite loci showed microsatellite instability in all TAs, as well as in normal squamous epithelium. p53 gene mutation was identified in 28.9% (11/38 TAs). All cases showed a common missense mutation in exon 8 at codon 273 (CGT-->CAT, Arg-->His), which was DNA contact mutation in the S10 beta strand. In association with microsatellite alterations, 7 of 9 TAs with p53 mutation in exon 8 at codon 273 also showed loss of heterozygosity (LOH) of p53 gene. LOH of p53 gene was detected in 83.8% (31/37 TAs). LOH at D2S123 on 2p16 near MSH2 gene and at D3S1611 on 3p22 near MLH1 gene was detected in 65.4% (17/26) and 71.4% (10/14) TAs, respectively. Frequencies of LOH at p53 and D2S123 were similar in non-cancerous areas and SCCs. LOH of p53 and D2S123 were found in 50% (5/10 TAs) of non-cancerous areas and 60% (9/15 TAs) of SCCs. Our results suggest that genetic instability induces esophageal tumor multicentricity, and that p53 gene contact mutation together with LOH are early events of the multistage carcinogenesis of multifocal primary esophageal SCC.     

Frequent loss of heterozygosity on chromosome 10p14-p15 in esophageal dysplasia and squamous cell carcinoma

High frequencies of loss of heterozygosity (LOH) on chromosome 10p14-p15 have been reported in various tumors, including glioma, pulmonary carcinoid and cervical, hepatic and prostatic carcinomas. These findings suggest the presence of a tumor suppressor gene at the loci. However, analysis of LOH on chromosome 10p14-p15 in esophageal tumors has not been reported. Therefore, we examined LOH on chromosome 10p14-p15 in 88 esophageal squamous cell carcinomas (SCC) (35 superficial- and 53 advanced-types) and 44 dysplasias by microsatellite assay. Five oligonucleotide primer sets for microsatellite loci D10S191, D10S501, D10S559, D10S558 and D10S249 were used. In dysplasias, frequent LOH was detected with markers D10S191 (26%) and D10S249 (33%). In superficial esophageal SCCs, frequent LOH was detected with markers D10S191 (26%), D10S559 (50%), D10S558 (29%) and D10S249 (33%). In advanced esophageal SCCs, we found frequent LOH was detected with markers D10S191 (38%), D10S501 (25%) and D10S559 (30%). There were no significant correlations between LOH on chromosome 10p14-p15 and clinicopathologic features, including patient age, sex, tumor location, depth of invasion and lymph node metastasis. These data suggest that a putative tumor suppressor gene for esophageal carcinogenesis may be located on chromosome 10p14-p15 and that malfunction of this gene may be involved in the development but not progression of esophageal tumors.     

Microsatellite alterations on chromosome 9 in chewing tobacco-induced oral squamous cell carcinomas from India

Genomic instability as reflected by microsatellite alterations in specific target regions is an important characteristic of oral squamous cell carcinoma (OSCC). Microsatellite instability (MSI) and loss of heterozygosity (LOH) on chromosome 9 has been reported as an early event in oral cancers, primarily from patients in the USA and UK. Hence, we examined 77 primary oral cancer tissues and corresponding peripheral blood cell (PBC) DNA from Indian oral cancer patients for LOH and MSI, using a panel of 11 microsatellite markers spanning chromosome 9 on p and q arms. The patients were long-time (minimum 10 years) tobacco chewers. The matched DNA samples were amplified by polymerase chain reaction, resolved on a denaturing polyacrylamide gel and visualized by silver staining. An overall of 62% (48/77 cases) of the patients demonstrated microsatellite alterations including 27% MSI and 52% LOH, although at individual loci MSI was observed in 3-8% patients and LOH in the informative cases ranged from 4 to 41%. A majority of the alterations occurred on the p arm at 9p21-23, with 85% (41/48 cases) genetic alterations concentrated between markers D9S157 and D9S161. Multiple alterations were seen in 56% (27/48) of the affected cases with 17 patients showing microsatellite alterations in three to eight loci. Our data show the incidence of genetic alterations primarily in the chromosomal region 9p21-23, and may be indicative of involvement of p16 (CDKN2) tumor suppressor gene on chromosome 9p21, in a subset of chewing tobacco-induced oral cancers.     

Clonal origin of lymph node metastases in bladder carcinoma

BACKGROUND: Evidence of genetic heterogeneity within urothelial carcinomas of the bladder has raised questions about the clonal origin of urothelial carcinoma and its metastases. High-grade urothelial carcinoma of the bladder frequently metastasizes to multiple regional lymph nodes in the pelvis. Whether or not these multiple lymph node metastases originate from the same tumor clone is uncertain. Molecular analysis of microsatellite alterations and X-chromosome inactivation status of distinct tumor cell populations from the same patient may further our understanding of the genetic basis of carcinoma progression and metastasis. METHODS: The authors examined 24 patients who underwent radical cystectomy for urothelial carcinoma. All patients had multiple (from two to four) lymph node metastases. Genomic DNA samples were prepared from formalin fixed, paraffin embedded tissue sections using laser-assisted microdissection. Loss of heterozygosity (LOH) assays for 3 microsatellite polymorphic markers on chromosome 9p21 (D9S171, region of putative tumor suppressor gene p16), 9q32 (D9S177, putative tumor suppressor gene involved in urothelial carcinoma tumorigenesis), and 17p13 (TP53, the p53 locus) were performed. In addition, X-chromosome inactivation analysis was performed in primary tumors and metastases from 10 female patients. RESULTS: In total, 79 tumors were analyzed. The overall frequency of allelic loss was 67% (16 of 24 tumors) in the primary urothelial carcinomas and 79% (19 of 24 tumors) in the metastatic carcinomas. The primary urothelial carcinoma showed LOH at the D9S171, D9S177, and TP53 loci in 39% (9 of 23 tumors), 30% (6 of 20 tumors), and 30% (7 of 23 tumors) of informative samples, respectively. LOH in > or = 1 lymph node metastases was seen at the D9S171, D9S177, and TP53 loci in 35% (8 of 23 tumors), 45% (9 of 20 tumors), and 48% (11 of 23 tumors) of informative samples, respectively. Eleven tumors demonstrated identical allelic loss patterns at all DNA loci both in the primary carcinoma and in all corresponding lymph node metastases. Three tumors showed allelic loss in the metastatic carcinoma but not in its matched primary carcinoma. Six tumors demonstrated a different LOH pattern in each of its lymph node metastases. Clonality analysis showed the same pattern of nonrandom X-chromosome inactivation both in the primary urothelial carcinoma and in all of the lymph node metastases in five of nine informative tumors studied. Four tumors showed a random pattern of X-chromosome inactivation in both the primary carcinoma and in the metastases. CONCLUSIONS: LOH and X-chromosome inactivation assays showed that multiple lymph node metastases and matched primary urothelial carcinomas of the bladder had the same clonal origin, suggesting that the capability for metastasis often arises in only a single clonal population in the primary tumor. The variable LOH patterns observed in some of the tumors likely reflect genetic divergence during the clonal evolution of urothelial carcinoma.     

Microsatellite instability and loss of heterozygosity in oral squamous cell carcinoma in Malaysian population

Loss of heterozygosity (LOH) and microsatellite instability (MSI) have been documented as important events in oral squamous cell carcinoma (OSCC). Five microsatellite markers D3S192, D3S966, D3S647, D3S1228 and D3S659 were selected on chromosome 3p because of high frequency of alterations reported in head and neck squamous cell carcinoma and the involvement of von Hippel Lindau (VHL) at 3p25-26 and the fragile histidine triad (FHIT) at 3p14.2 genes proven in many tumour types. A total of 50 archival tissue samples of OSCC and corresponding normal samples were analyzed for LOH and MSI status. The overall LOH for the markers selected on 3p was 56 out of 189 informative cases (29.6%). The most frequent LOH was identified for the marker D3S966 which was 18/42 (42.8%) of informative cases suggesting the presence of putative tumour suppressor genes (TSGs) in this loci. In this study, high frequency of microsatellite instability was found in D3S966 which was 28.6% of informative cases; this reveals the possibility of mutations of MMR genes in this region. Frequent microsatellite alterations (MA) were observed in 3 markers D3S966 (71.4%), D3S1228 (56.7%) and D3S192 (41.0%). There was no significant association between LOH with gender, tumour stages and differentiation grades. However, there was a significant association between tumour stage and differentiation grades with MSI status in OSCC in Malaysian population with p values of 0.002 and 0.035, respectively. There was also a significant association between MA and differentiation grades (p=0.041).     

Multiple high-grade bronchial dysplasia and squamous cell carcinoma: concordant and discordant mutations.

Loss of heterozygosity (LOH) involving chromosomes 3p, 5q, 9p, or 17p and aberrant expression or mutation of p53 are reported previously in selected bronchial dysplasias and squamous cell cancers (SCCs). Yet, comprehensive analyses of LOH patterns at these chromosomal sites and of p53 alterations are not reported for histologically normal bronchial epithelium, high-grade bronchial dysplasia, and SCC present in the same pulmonary resections. Whether concordant or discordant genetic changes are detected in these bronchial tissues, especially when multiple high-grade dysplastic bronchial lesions are present, was studied. Genomic DNA was microdissected from eight pulmonary SCCs and high-grade dysplastic lesions that were associated with SCC. In four cases, two independent high-grade dysplastic bronchial lesions were identified. When available, histologically normal bronchial epithelium was microdissected. Germ-line genomic DNA was isolated from normal lymph nodes. LOH was assessed for 15 microsatellite markers on chromosomes 3p, 5q, 9p, or 17p, sites frequently deleted in lung cancers. Immunohistochemical p53 expression was studied and correlated with p53 DNA sequence analyses. Progressive LOH for these markers was found when SCCs were compared with high-grade dysplasia and histologically normal bronchial epithelium present in the same resections. Histologically normal bronchial specimens had LOH in up to 27% of informative markers. High-grade dysplastic lesions exhibited LOH for 18-45% and SCC had LOH for 18-73% of the markers. Common regions of LOH were found in some dysplasias compared with SCCs. In other dysplasias, discordance was found relative to SCCs, especially for p53 mutations. In cases with a single or second high-grade dysplasia associated with SCC, heterogeneity in LOH markers was detected. These concordant and discordant changes were consistent with convergent and divergent clonal selection pathways in pulmonary squamous cell carcinogenesis. Some histologically normal bronchial epithelial tissues had genetic changes more similar to those in the SCCs than in dysplastic lesions. DNA loss or mutations accumulate in SCC, but discordant genetic changes can exist in the same carcinogen-exposed bronchial tissues. These findings have implications for lung cancer prevention trials.     

Analysis of genetic alterations associated with DNA diploidy, aneuploidy and multiploidy in gastric cancers

OBJECTIVE: Recent studies have shown a close association between DNA ploidy status (diploidy, aneuploidy and multiploidy) identified by the crypt isolation technique and specific genetic alterations in colorectal carcinomas. However, such an association has not been elucidated for gastric tumors, even though they share common genetic features with colorectal carcinomas. In the present study, we established an association between DNA ploidy status and genetic alterations in gastric cancer. METHOD: The DNA ploidy status of gastric tumors was classified as diploid, aneuploid or multiploid using the crypt isolation technique, which allows isolation of pure tumor crypt from tumor tissue. Crypt isolation combined with DNA cytometric sorting, polymerase chain reaction assay using 26 microsatellite markers and direct sequencing of the p53 gene were used to detect allelic imbalances [loss of heterozygosity (LOH) or allelic loss], microsatellite imbalance (MSI) and mutation of p53 in 54 gastric cancers (13 diploid, 12 aneuploid, 29 multiploid). RESULT: Diploid tumors showed few genetic alterations, including allelic imbalances and p53 mutations. In contrast, aneuploid tumors and multiploid tumors (in particular, aneuploid populations of multiploid tumors) exhibited multiple genetic alterations, including allelic imbalances and p53 mutations. In addition, the frequencies of genetic alterations observed in the corresponding diploid fractions of multiploid tumors were relatively higher than in diploid tumors. MSI was commonly observed in diploid, aneuploid and multiploid carcinomas. CONCLUSIONS: The present results indicate that in gastric carcinomas, diploid tumors are generally non-LOH and MSI, whereas aneuploid and multiploid tumors are associated with LOH and MSI, suggesting that the genetic profile of these carcinomas is dependent on the tumor's ploidy status.     

Heterogeneity of allelic deletions within melanoma metastases

During the initiation and progression of malignant melanoma, a series of different genetic events accumulate on several different chromosomes. The biological heterogeneity of tumour cells presents a major problem, preventing effective treatment of melanoma. To examine the degree of genetic heterogeneity, we searched for allelic losses (loss of heterozygosity; LOH) on chromosomes 9p, 9q, 1p and 17p, examining different areas within human melanoma metastases. All of the examined metastases were informative within at least one dissected area for at least one marker. Out of 29 areas in 11 melanoma metastases, 58% showed LOH with at least one marker. On chromosome 9p21-22, eight out of 26 informative loci (31%) showed LOH at D9S171 (three not informative), two out of 18 (11%) at IFNA (11 not informative) and seven out of 24 (29%) at D9S169 (five not informative). LOH on chromosome 9q22.3 was examined by the microsatellite marker D9S12; three out of 24 areas (12.5%) showed LOH, and five were not informative. Deletions on chromosome 1p were assessed using D1S450. Four out of 25 (16%) showed LOH; four were not informative. Deletions on chromosome 17p13 were examined with TP53; two out of 21 cases (9%) showed LOH, and eight were not informative. Our data demonstrate an impressive heterogeneity of allelic losses in the investigated chromosomal areas within the same metastatic lesion. This suggests that there is not one specific genetic alteration that accounts for melanoma progression to metastases. Rather there seem to be multiple genetic alterations accumulating even on the same chromosome, and progression from melanoma to metastases is paralleled by the accumulation of clones harbouring multiple genetic abnormalities.     

Microsatellite DNA analysis does not distinguish malignant from benign pleural effusions

Distinguishing malignant from benign pleural effusions using routine cytology is a common diagnostic problem. Recently, genetic alterations, including microsatellite instability (MSI) and loss of heterozygosity (LOH), have been described in malignant pleural effusions and proposed as methods improving diagnostics. The purpose of this study was to evaluate a panel of molecular markers for the detection of genetic alterations of cells in pleural effusions and to determine their diagnostic value as an additional test to cytologic examination. Pleural fluid and peripheral blood from 48 patients (36 male and 12 female, median age 71 years) were analyzed. Twenty-six patients had malignant pleural effusion, including 23 lung cancer and three metastatic non-pulmonary carcinoma. The control group consisted of 22 patients with benign pleural effusions. Only 14 malignancy-associated pleural effusions were cytology-positive for malignant cells (54%), whereas all benign pleural effusions were negative. DNA was extracted from all the samples and analysed for MSI and/or LOH using the following microsatellite markers: D3S1234, D9S171, D12S363, D17S250, D5S346 and TP53Alu, located at five chromosomal regions: 3p, 9p, 12q, 17q, 5q. Microsatellite analysis of the pleural fluid pellet exhibited genetic alterations in two neoplastic pleural fluid cases and in one inflammatory case. Two out of 26 (7.6%) patients with malignant pleural effusion showed genetic alterations. One exhibited MSI in three different microsatellite markers (D17S250, D9S171, D3S134) and the other showed LOH in marker D3S134. One out of 22 (4.5%) patients with benign pleural effusion showed LOH in marker D3S134. In conclusion, genetic alterations at the level of microsatellite DNA, were detected only in very few cases of malignant pleural effusions, and in one case of benign pleural effusion. Thus, our data suggest that microsatellite DNA analysis does not facilitate the diagnosis of malignant pleural effusion.     

Papillary lesions of the breast: a molecular progression?

Summary Introduction. Breast papillary lesions represent a heterogeneous group of tumors ranging from benign to malignant, including several intermediate forms. Malignant papillary tumors are rare and their molecular characterization is still limited. A few studies pointed to the presence of specific genetic alterations that could be relevant both for diagnostic purposes and to elucidate tumour development and progression. In order to look into the issue, we compared LOH relative frequencies of four microsatellite markers located on chromosome 16 in a set of morphologically different papillary breast lesions. LOH at TP53 locus was also analyzed throughout lesions. Materials and methods. Fifteen cases were analyzed. Sections including a malignant papillary lesion, a benign lesion (when available), and normal breast tissue were selected. Fifteen malignant and twelve benign areas were microdissected using the Leica laser microdissection system (AS LMD). After DNA extraction samples were tested for the following markers: TP53, D16S423, D16S310, DS163210 and D16S476, and analyzed on ABI PRISM 3100 (Applied Biosystems, Foster city CA). Results. Fourteen malignant lesions and twelve paired benign areas appeared to be informative for at least one of the four markers on chromosome 16. In particular, LOH at loci 16p13 and 16q21 was detected in both benign and malignant lesions, whereas LOH at locus 16q23 was limited to malignant lesions. Nine malignant and seven benign lesions were informative for LOH at TP53 locus, that was found to be significantly associated (p=0.01) with the malignant phenotype. Conclusions. Our data suggest an involvement of chromosome 16 mutations in the early steps of breast papillary tumorigenesis. TP53 deletion and possibly LOH at 16q23 appear to play a role as progression factors, being they significantly associated with malignant transformation of breast papilloma.     

Loss of heterozygosity and microsatellite instability in ductal carcinoma in situ of the breast

To investigate the alterations of genetic instabilities in carcinogenesis of the breast, we analyzed the allelotypic profile of 65 ductal carcinomas in situ (DCIS), compared with that of 207 invasive ductal carcinomas (IDC) of the breast. These studies were performed by means of examining microsatellite-length polymorphisms at seven loci (AluVpa, ESR, D11S988, D13S267, D16S398, D17S1159, and D17S855) from microdissected paraffin sections. Allelic loss or imbalance, considered a loss of heterozygosity (LOH), tended to be more frequently seen in IDC than in DCIS. In particular, the frequency of LOH at the 17p locus was significantly higher in IDC than in DCIS (42 vs. 23%, P=0.022). LOH in DCIS was most frequently seen at D16S398 (26%). LOH frequency at D16S398 in low- and intermediate-grade DCIS was higher than that in high-grade DCIS, while LOH frequencies at D11S988 and D17S1159 in low- and intermediate-grade DCIS was lower than those in high-grade DCIS. LOH frequency at D11S988 in non-comedo type DCIS was lower than that in comedo type DCIS. Furthermore, the frequency of microsatellite instability (MSI) at only one locus in DCIS (28%) was statistically higher than that in IDC (6%) (P<0.001), while there was no difference between the frequency of MSI at multiple loci in DCIS (6%) and that in IDC (3%). Together, these observations indicate that chromosomal losses of 16q may occur in low- and intermediate-grade DCIS and those of 11p and 17p may occur high-grade DCIS, and that MSI occurring at only one locus is not yet clear and MSI at multiple loci is uncommon in not only IDC but also DCIS of the breast.     

Microsatellite alterations in head and neck squamous cell carcinoma and relation to expression of pimonidazole, CA IX and GLUT-1.

BACKGROUND AND PURPOSE: Because the locoregional control for HNSCC is still disappointing, research efforts focus on the exploration of new molecular markers located in both tumour and microenvironment, which could help stratify patients. The aim of the present work was therefore first to assess microsatellite alterations and hypoxia in HNSCC as possible molecular markers. Second, a relation between both was investigated, as hypoxia is known to select for genetic alterations. MATERIAL AND METHODS: Forty-eight patients with advanced HNSCC treated by surgery+/-radiotherapy were included. MSI and LOH were investigated with microsatellite markers using automatic fragment analysis. The presence of hypoxia was assessed by immunohistochemistry for pimonidazole, CA IX and GLUT-1. The mutual relationship between MSI/LOH and hypoxia was evaluated. RESULTS: No MSI was detected in this patient group. LOH occurred mostly on chromosomal arms 3p, 5q, 9p, 17p and 17q. Patients with LOH at D17S799, located in the near environment of p53, showed a higher CA IX expression (p=0.01). CONCLUSIONS: LOH is a possible molecular marker in HNSCC. The positive correlation between LOH at D17S799 and CA IX is in full concordance with previous publications linking hypoxia to selective pressure on the p53 gene.     

p53 mutation and allelic loss of chromosome 3p, 9p of preneoplastic lesions in patients with nonsmall cell lung carcinoma

BACKGROUND: An accumulation of mutations can result in carcinogenesis. Comparing genetic alterations in preneoplastic lesions with those seen in cancer in the same patient may be helpful in the early diagnosis of lung carcinoma or preneoplastic lesions. METHODS: To identify genetic alterations that may play a role in the development of nonsmall cell lung carcinoma (NSCLC), the authors examined the p53 gene and microsatellite markers on chromosome 3p (D3S643, D3S1317), 9p (D9S171, IFNA) in 35 bronchial metaplastic lesions and 28 alveolar hyperplastic lesions from 61 patients. RESULTS: A total of 8 metaplastic lesions (1 squamous metaplasia and 7 dysplasias) and 3 alveolar hyperplastic lesions (with atypia) showed genetic alterations, including loss of heterozygosity (LOH) of 3p, 9p and mutations of the p53 gene. In an analysis of microsatellite markers, 5 of 35 cases of squamous cell carcinoma (SCC) and 3 of 26 cases of adenocarcinoma (Ad) showed LOH in both preneoplastic lesions and synchronous cancers. Nine patients (25.7%) with SCC and 6 patients (23.1%) with Ad were shown to have mutations of the p53 gene by single-strand conformation polymorphism. In 2 of these 9 patients with SCC, the same mutation was observed in both dysplasia and SCC. CONCLUSIONS: These findings suggest that several genetic alterations may occur in preneoplastic lesions or the early stage of SCC of the lung, whereas the genetic alterations examined appeared to occur relatively late in the pathogenesis of pulmonary adenocarcinoma.     

Investigation of allelic imbalances on chromosome 3p in nasopharyngeal carcinoma in Tunisia: high frequency of microsatellite instability in patients with early-onset of the disease

Tunisia is one of the world's intermediate risk areas for nasopharyngeal carcinoma (NPC). Loss of heterozygosity (LOH) on the short arm of chromosome 3 (3p) is the most frequent genetic change reported in NPC from endemic areas. In the present study, we investigate the incidence of LOH and microsatellite instability (MSI) on chromosome 3p in 49 microdissected primary NPC specimens and corresponding non-cancerous tissues from Tunisian patients using six microsatellite polymorphic markers. LOH at one or more markers was observed in 40 out of 48 informative cases (83.3%). The markers D3S1038 at 3p25.2-26.1 and D3S1076 at 3p21.1-21.2 have showed the highest frequency of LOH (51.3%), followed by D3S1067 at 3p14.3-21.1 (48.7%), D3S1568 at 3p21.3 (47.4%), D3S659 at 3p13 (15.3%), and D3S1228 at 3p14.1-14.2 (11%). Interestingly, MSI at one or more microsatellite markers was observed in 15 cases (31.2%). The highest frequency of MSI was presented by D3S1568 (18.4%), D3S1067 (17.9%), and D3S1038 (12.8%). With regard to clinicopathological features, LOH was found to be less common in young patients (under 25 years) than in adults (p=0.04), whereas MSI was found to be more frequent in patients under 45 years than in older patients (p=0.006). No significant correlation was found between LOH or MSI and the other clinicopathological features investigated including, gender, tumor size, lymph node metastasis, UICC clinical stage, and histological subtype. This study revealed different patterns of allelic imbalance on chromosome 3P in NPC between age groups in Tunisia, and suggests an alteration in the DNA mismatch repair machinery that may be, in part, responsible of the early age onset form of this disease in North African populations. More attention should be given to the mismatch repair system in the juvenile form of this disease in future studies.     

Genetic alterations in gastric cancer cell lines and their original tissues

To investigate the genetic changes that occur during establishment of gastric cancer cell lines, 4 gastric cancer cell lines and their original tumor tissues were examined for microsatellite instability (MSI), loss of heterozygosity (LOH), and p53 mutation. MSI status did not change during the establishment, though the cell lines gained frameshift mutation in some of the genes with polytract coding sequences. There was no difference in p53 mutation between the cell lines and the original tumors. The frequency of LOH was similar between the cell lines and each original tumor, in the range 30.8% to 85.7%. The difference in the LOH results between the cell lines and the original tumors was not greater than the difference between the different areas of the original tumors. The above results suggested that most of the genetic alterations, such as MSI, LOH, and p53 gene mutation, were sustained during the establishment of gastric cancer cell lines. Minor genetic differences between original tumor tissues and cancer cell lines could be explained as a tumor heterogeneity because separate areas of the original tumor tissues manifest similar variations.     

肝细胞癌8号染色体微卫星变异的研究

目的　　探讨肝细胞癌(HCC)8号染色体微卫星变异的特点及其与临床病理的相关性。方法采用MegaBACE　500型自动化DNA分析系统,对56例HCC中8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 在8 号染色体上10 个基因座发生LOH 的总频率为66.1%(37 /56),MSI 的频率为12.5%(7 /56),AI的频率为19.6%(11/56)。LOH以D8S261最高为53.5%(23/43),其次为D8S1721(52.5%)和D8S1771(52.5%)。D8S277基因座,血清HBsAg阳性患者的LOH频率显著高于HBsAg阴性者(P<0.01),D8S261、D8S298和D8S1733基因座,血清HBsAg阴性患者的LOH频率显著高于HBsAg阳性者(P<0.01);D8S298和D8S1771基因座,肿瘤直径>3cm的LOH率明显高于￣<3cm组(P<0.05和P<0.01);在D8S1721基因座,无包膜或包膜不完整的肿瘤的LOH显著高于包膜完整的肿瘤(P<0.01);D8S298和D8S1771基因座,肝内转移者的LOH明显高于无肝内转移者(P<0.05)。MSI和AI与HCC临床病理学特点无明显相关性。结论　　HCC的8号染色体上存在广泛的微卫星变异,其中以代表肿瘤抑制基因路径的LOH方式在HCC的发生和发展过程中起重要作用,代表错配修复基因路径的MSI的作用次之。特定基因座的LOH与临床和病理学参数有一定的相关性。     

The presence of candidate tumor suppressor gene loci at chromosome 3p for oral squamous cell carcinomas

We investigated the short arm of chromosome 3 (3p) for allelic imbalances, including loss of heterozygosity (LOH) and microsatellite instability (MSI) in 40 primary oral squamous cell carcinomas (SCCs) using 10 microsatellite markers and constructed a deletion map for this chromosome arm. We examined 40 primary tumor tissues, 40 corresponding normal tissues, and seven lymph node metastatic tissues. LOH at one or more loci was found in 24/40 (60%) of tumors. Deletion mapping of these tumors revealed at least three discrete, commonly deleted regions on the chromosome arm. Furthermore, we detected MSI in six of those tested cases (15%). We compared our results with the clinicopathologic features. A number of sites displaying LOH at 3p could be detected in early stage lesions, and the frequencies of LOH tended to be higher in later clinical stages. Thus, the frequent LOH was observed from early stage in pTNM classification. An unknown tumor suppressor gene in the genesis of oral squamous cell carcinoma may exist in 3p.     

Microsatellite instability in esophageal adenocarcinoma

The frequency of microsatellite instability (MSI), a result of defective mismatch repair during DNA replication, has been reported inconsistently in primary esophageal adenocarcinoma (EADC). Using a panel of 15 markers, the primary aim of this study was to analyze the frequency of MSI in a well-characterized series of 27 primary EADCs, defined according to strict clinicopathologic criteria. Polymerase chain reaction was used to amplify the following microsatellite repeat loci: D2S123, D10S197, D2S119, D11S904, D2S147, D3S1764, D7S1830, D7S1805, D2S434, D9S299, BAT25, BAT26, D5S346, D17S250, and TGF-beta-RII. Tumors were classified as microsatellite-stable (MSS) when no alterations were seen in tumor DNA compared to matched normal tissues, low-level MSI (MSI-L) when 1-5 of 15 markers were altered, and high-level MSI (MSI-H) when more than five markers were altered. Using these stringent criteria, 9/27 (33%) tumors were MSS, 18/27 (67%) tumors were MSI-L, and no tumor was MSI-H. Immunohistochemistry demonstrated cell nuclear expression of DNA mismatch repair proteins (both hMLH1 and hMSH2) in 78% (21/27) of tumors. No associations were seen between MSI and immunohistochemical expression of hMLH1, hMSH2, alterations in p53 or MBD4, tumor grade, pathologic stage, or patient survival. In conclusion, the finding of low levels of MSI in most tumors suggests an inherent baseline genomic instability, and potentially increased susceptibility to mutations during the progression of esophageal adenocarcinoma.