Combined LOH/CGH analysis proves the existence of interstitial 3p deletions in renal cell carcinoma

We have recently developed an allele titration assay (ATA) to assess the sensitivity and influence of normal cell admixture in loss of heterozygosity (LOH) studies based on CA-repeat. The assay showed that these studies are biased by the size-dependent differential sensitivity of allele detection. Based on these data, we have set up new criteria for evaluation of LOH. By combining these new rules with comparative genome hybridization (CGH) we have shown the presence of interstitial deletions in renal cell carcinoma (RCC) biopsies and cell lines. At least three out of 11 analysed RCC cell lines and three out of 37 biopsies contain interstitial deletions on chromosome 3. Our study suggests the presence of several regions on human chromosome 3 that might contribute to tumor development by their loss: (i) 3p25-p26, around the VHL gene (D3S1317); (ii) 3p21. 3-p22 (between D3S1260 and D3S1611); (iii) 3p21.2 (around D3S1235 and D3S1289); (iv) 3p13-p14 (around D3S1312 and D3S1285). For the first time, AP20 region (3p21.3-p22) was carefully tested for LOH in RCC. It was found that the AP20 region is the most frequently affected area. Our data also suggest that another tumor suppressor gene is located near the VHL gene in 3p25-p26.     

Discovery of frequent homozygous deletions in chromosome 3p21.3 LUCA and AP20 regions in renal, lung and breast carcinomas

We searched for chromosome 3p homo- and hemizygous losses in 23 lung cancer cell lines, 53 renal cell and 22 breast carcinoma biopsies using 31 microsatellite markers located in frequently deleted 3p regions. In addition, two sequence-tagged site markers (NLJ-003 and NL3-001) located in the Alu-PCR clone 20 region (AP20) and lung cancer (LUCA) regions, respectively, were used for quantitative real-time PCR (QPCR). We found frequent (10-18%) homozygous deletions (HDs) in both 3p21.3 regions in the biopsies and lung cancer cell lines. In addition, we discovered that amplification of 3p is a very common (15-42.5%) event in these cancers and probably in other epithelial malignancies. QPCR showed that aberrations of either NLJ-003 or NL3-001 were detected in more than 90% of all studied cases. HDs were frequently detected simultaneously both in NLJ-003 or NL3-001 loci in the same tumour (P<3-10(-7)). This observation suggests that tumour suppressor genes (TSG) in these regions could have a synergistic effect. The exceptionally high frequency of chromosome aberrations in NLJ-003 and NL3-001 loci suggests that multiple TSG(s) involved in different malignancies are located very near to these markers. Precise mapping of 15 independent HDs in the LUCA region allowed us to establish the smallest HD region in 3p21.3C located between D3S1568 (CACNA2D2 gene) and D3S4604 (SEMA3F gene). This region contains 17 genes. Mapping of 19 HDs in the AP20 region resulted in the localization of the minimal region to the interval flanked by D3S1298 and D3S3623 markers. Only four genes were discovered in this interval, namely, APRG1, ITGA9, HYA22 and VILL.     

Deletion mapping using quantitative real-time PCR identifies two distinct 3p21.3 regions affected in most cervical carcinomas

We report chromosome 3p deletion mapping of 32 cervical carcinoma (CC) biopsies using 26 microsatellite markers located in frequently deleted 3p regions to detect loss of heterozygosity and homozygous loss. In addition, two STS markers (NLJ-003 and NL3-001) located in the 3p21.3 telomeric (3p21.3T) and 3p21.3 centromeric (3p21.3C) regions, respectively, were used for quantitative real-time PCR as TaqMan probes. We show that quantitative real-time PCR is reliable and sensitive and allows discriminating between 0, 1 and 2 marker copies per human genome. For the first time, frequent (five of 32 cases, i.e. 15.6%) homozygous deletions were demonstrated in CCs in both 3p21.3T and 3p21.3C regions. The smallest region homozygously deleted in 3p21.3C was located between D3S1568 (CACNA2D2 gene) and D3S4604 (SEMA3F gene) and contains 17 genes previously defined as lung cancer candidate Tumor suppressor genes (TSG(s)). The smallest region homozygously deleted in 3p21.3T was flanked by D3S1298 and NL1-024 (D3S4285), excluding DLEC1 and MYD88 as candidate TSGs involved in cervical carcinogenesis. Overall, this region contains five potential candidates, namely GOLGA4, APRG1, ITGA9, HYA22 and VILL, which need to be analysed. The data showed that aberrations of either NLJ-003 or NL3-001 were detected in 29 cases (90.6%) and most likely have a synergistic effect (P<0.01). The study also demonstrated that aberrations in 3p21.3 were complex and in addition to deletions, may involve gene amplification as well. The results strongly suggest that 3p21.3T and 3p21.3C regions harbor genes involved in the origin and/or development of CCs and imply that those genes might be multiple TSG(s).     

Frequent loss of heterozygosity in early non-small cell lung cancers at chromosome 9p21 proximal to the CDKN2a gene

Deletions involving the chromosome 9p21 region have been reported as frequent events in non-small cell lung cancer (NSCLC). To investigate potential tumor-suppressor gene (TSG) loci within the 9p21 region, which also harbors the candidate TSG locus CDKN2a, we are studied 32 cases of primary NSCLC for loss of heterozygosity (LOH). Tumor and paired normal lung cells were microdissected from lung tissue imprints and all samples screened using PCR-LOH analysis with 15 9p markers. In addition, 3 NSCLC cell lines and their matched normal lung and tumor DNA were similarly analyzed. LOH at the marker D9S259, which is proximal to the CDKN2a locus, was found most frequently (52%) while LOH at D9S942, the marker closest (5 kb) to the CDKN2a gene, was seen in only 17%. Homozygous loss of markers close to CDKN2a gene was, however, detected in 2 of the cell lines and one accompanying tumor sample. We propose that a TSG in the region of deletion proximal to the CDKN2a gene within 9p21 may play a significant role in the pathogenesis and progression of NSCLC. Int. J. Cancer 71:213–217, 1997. © 1997 Wiley-Liss, Inc.     

Loss of heterozygosity at 3p14.2 in clear cell renal cell carcinoma is an early event and is highly localized to the FHIT gene locus

The VHL tumor suppressor gene (TSG) at 3p25-26 is strongly implicated in the pathogenesis of clear cell renal cell carcinoma (cRCC). In addition, 3p14.2 and 3p21 are suspected of harboring additional TSGs in cRCC, with FHIT being a candidate TSG at 3p14.2. We examined 87 microdissected, histologically well-defined cRCCs classified according to tumor-node-metastasis (TNM) stage (stage 1, 23 cases; stage 2, 14 cases; stage 3, 24 cases; stage 4, 26 cases) and Fuhrman grade (grade 1, 24 cases; grade 2, 19 cases; grade 3, 19 cases; grade 4, 8 cases; sarcomatoid cRCC, 17 cases) for loss of heterozygosity (LOH) at 3p14.2 and 3p25-26 using a series of precisely mapped microsatellite probes. We found that LOH at 3p14.2 exceeded LOH at 3p25-26 in frequency (69% versus 48.3%; P < 0.03) and was highly localized to markers within the FHIT gene locus (D3S1300 and D3S4260), with the majority of chromosomal breakpoints also mapping to this region. In addition, 3p14.2 LOH (P < 0.03), but not 3p25-26 LOH (P = nonsignificant), was associated with lower tumor grades (grades 1-3). These findings suggest that 3p14.2 genomic deletions may be among the earliest events in cRCC pathogenesis, preceding genomic deletions at the VHL locus. FHIT, or an as yet undiscovered TSG mapping to the D3S4103-D3S4260 interval, could be the molecular target of the 3p14.2 deletions.     

Loss of 14q31-q32.2 in renal cell carcinoma is associated with high malignancy grade and poor survival

The present study was undertaken to further approach the importance of 14q deletions in renal cell carcinoma (RCC) development. The initial screening using 2 RFLP markers from distal 14q identified loss of heterozygosity (LOH) in 17 of 45 informative cases (38%). In addition, in 37 patients with primary RCCs, it was shown that cases with LOH at D14S1 had significantly shorter survival as compared to cases with-out LOH (p<0.005). Subsequently, 19 primary tumors and 6 metastases were genotyped for 20 polymorphic markers and the findings were evaluated in relation to the clinical characteristics of the primary tumor and the survival during follow-up. Overall LOH was identified in 11 of the primary tumors (58%) and 4 of the metastases (66%). In metastases as well as in primary tumors the highest frequency of LOH was detected with markers from the distal part of the chromosome i.e., 14q32. Five minimal regions of overlapping deletions were identified, three of which (II, IV and V) were defined from the primary RCCs. From centromere to telomere these include region I proximal of D14S259, region II between D14S255 and D14S588, region III in the D14S61-D14S617 interval, region IV between D14S617 and D14S260, and region V telomeric of D14S1007. For the primary tumors, losses in regions IV and V were each significantly associated with high tumor grade (i.e., grade 3; p<0.05). Furthermore, LOH within region IV was also associated with a significantly shorter survival (p=0.02). In conclusion, the high frequency of distal 14q LOH supports the relevance of this alteration for the development of RCC.     

Allele loss on chromosome-3 in squamous-cell carcinoma of the head and neck correlates with poor clinical prognostic indicators

Cytogenetic studies in squamous cell carcinoma of the head and neck (SCCHN) have identified a clustering of breakpoints in a number of chromosomes, including chromosome 3. We have undertaken a loss of heterozygosity analysis (LOH) of 36 SCCHN and six solid tumours which were not squamous cell, and their respective normal specimens, using a bank of microsatellite markers, with the aim of identifying specific sites of frequent loss on chromosome 3. The analysis was undertaken with 12 microsatellite markers, 10 of which are on the p arm of chromosome 3. Allelic loss greater than 10% was seen at four sites; D3S1269 (13%), D3S1079 (23%), D3S659 (23%) and D3S1293 (31%). None of this series of tumours showed loss of the whole chromosome, however 47% of the tumours analysed had LOH at one or more loci. The highest incidence of LOH was found at D3S1293 in the 3p24-p25 region. The second highest region with LOH was found at D3S1079 and D3S659 at 3p13. The remaining markers telomeric and centromeric to these two regions were found to have a LOH of less than 10%. Furthermore, we found a strong association between loss of one marker on chromosome 3 in these SCCHN and poor clinical prognostic indicators; such as site, pathological differentiation, positive nodes at pathology (p<0.05) and TNM status (p<0.05). This study has identified two regions in SCCHN that are most likely to be important in the development of squamous cell carcinoma of the head and neck at 3p24-p25 and 3p13 and may indicate sites of novel tumour suppressor genes in this disease.     

Loss of heterozygosity on chromosome arms 3p and 6q in microdissected adenocarcinomas of the uterine cervix and adenocarcinoma in situ

BACKGROUND: Despite the increasing frequency of adenocarcinomas of the uterine cervix, little is known regarding inactivation of tumor suppressor genes (TSGs) in this tumor type. The authors analyzed loss of heterozygosity (LOH) in 36 carcinomas of the cervix with glandular differentiation, and 5 adenocarcinoma in situ in 40 patients. METHODS: The authors analyzed samples using laser capture microdissection from archival material and DNA amplified with microsatellite markers on the following loci: 3p14.2 (D3S1234, D3S1300), 3p21.3 (D3S1029, D3S1447), 3p22-24 (D3S1537, D3S1351), 6q21-23.3 (D6S250), 6q25.1 (ESR), 6q25.2 (D6S255), 8p21 (D8S136, D8S1820), 13q12.3 (D13S220, D13S267), 17q21 (D17S579, D17S855). Eight additional markers spanning the short arm of chromosome 3 (3p12-p25) and six spanning the long arm of chromosome 6 (6q11-q27) were studied in the cases showing LOH to further define the deletion intervals. RESULTS: The frequency of allelic loss in cancers was chromosome 3p: 49% (p14.2: 35%, p21.3: 23%, p22-24: 41%), 6q: 48% (q21-23.1: 39%, q25.1: 45%, q25.2: 7%), 13q: 22%, 17q: 6%, and 8p: 18%. On chromosome arm 3p, the authors' data suggest at least two discrete areas of deletion: a proximal area between markers D3S1234 (p12) and D3S1766 (p14.2-14.3), and a second distal interval, telomeric from marker D3S4623 (p21.3). On chromosome 6q, the deletion area is between marker D6S300 (q22) and D6S255 (q25.2). Two of five preneoplastic lesions showed LOH on chromosome arm 3p, and two five showed allelic loss on chromosome arm on 6q, suggesting the genes might be inactivated early in cervical tumorigenesis. CONCLUSIONS: The authors have identified three chromosomal regions that may harbor TSGs involved in the development/progression of adenocarcinomas of the uterine cervix, 3p12-14.2, 3p21.3-pter, and 6q22-25.2. Deletions also were detected in adenocarcinoma in situ, suggesting the genes may be inactivated early in cervical tumorigenesis.     

Deletions at chromosome 2q and 12p are early and frequent molecular alterations in bronchial epithelium and NSCLC of long-term smokers

Most lung cancer is attributed to long-term smoking. In order to define chromosomal regions with an accumulation of smoking-related early molecular damage, we applied 15 microsatellite markers at 8 chromosomal regions (2q35-q36, 3p21.3, 3p14.2, 3p25, 10q11.2, 11p14-15, 12p13.1-p12.3 and 12q14) in an allelotyping study. We studied samples of 42 patients with primary non-small cell lung cancer (NSCLC) (25 squamous cell carcinomas, 13 adenocarcinomas, 2 large cell and 2 bronchioalveolar carcinomas) to compare the frequency of allelic loss in cancer tissue of smokers with matched bronchial epithelium. As a control group we used 11 samples of non-smokers. In NSCLC we found significantly higher frequencies of loss of heterozygosity (LOH) than in matched tumor free bronchial epithelium (p = 0.007). Most frequently, allelic loss was detected in NSCLC at chromosome 3p [3p25 (46%), 3p21.3 (45%), 3p14 (40%)], at 2q35 (24%), 12p12 (29%) and 12q14 (13%), but infrequently at 10q11 (7%) and 11p14-15 (5%). In corresponding histological normal bronchial epithelium, the highest percentage of LOH was found at chromosome 3p [3p21 (17%), 3p25 (12%), 3p14 (9%)] and chromosome 2q (2q35-q36) (17%) and 12p (12p12-p13) (12%). LOH in histologically normal bronchial epithelium was significantly associated with long-term smoking (p = 0.048), especially at chromosome 12p12 (p = 0.018). Our results demonstrate two further deletion hot spots at the chromosomal region 2q35-q36 and 12p12-p13 in tumor tissue of NSCLC and matched histological normal bronchial epithelium of long-term smokers, reflecting a phenomenon referred to as 'field cancerization'. These chromosomal regions represent interesting loci for potential NSCLC associated tumor suppressor genes and could be useful as screening markers for molecular risk assessment of smokers.     

Loss of heterozygosity at loci of candidate tumor suppressor genes in microdissected primary non-small cell lung cancer

To investigate the etiological association of allelic loss at chromosomal regions containing tumor suppressor genes (TSGs) in non-small cell lung cancer (NSCLC) in Taiwan, we examined 48 microdissected NSCLC samples for loss of heterozygosity (LOH) at nine loci where TSGs are localized nearby. The associations of LOH at each locus with clinicoparameters and prognosis were also examined. The frequent LOH was observed using markers, D3S1285 near the FHIT gene (58.3%), D17S938 near the p53 gene (56.7%), D9S925 near the p16 gene (54.5%), and D13S153 near the RB gene (47.6%). The occurrence of LOH at each TSG locus was compared with the patients' clinicoparameters. The incidence of LOH at D17S938 (p53 gene) and D3S4545 (VHL gene) was significantly higher in squamous carcinoma tumors than in adenocarcinoma tumors (P = 0.003 and 0.024, respectively). LOH of these two loci also occurred frequently in tumors from smoker patients compared to that from nonsmoker patients (P = 0.013 and 0.025, respectively). LOH at D13S153 (RB gene) was also associated with smoking (P = 0.008). In addition, the prognostic analyses indicated that the patients with LOH at D18S535 (18q21, near the SMAD2/4 gene) had significantly longer post-operative survival time compared to those without LOH (P = 0.03). Our results suggested that LOH at FHIT, p53, and p16 genes may occur frequently in NSCLC patients in Taiwan. In addition, LOH at p53, RB, and VHL may associate with smoking or squamous carcinoma patients and LOH at SMAD2/4 may be correlated with better prognosis.     

Prognostic implications of loss of heterozygosity at 8p21 and 9p21 in head and neck squamous cell carcinoma

Loss of heterozygosity (LOH) in chromosomal regions that harbor tumor suppressor genes from tumor tissue may lead to decreased survival time in cancer patients with squamous cell carcinoma of the head and neck (HNSCC). We studied 8 regions frequently lost in HNSCC in 150 patients having a primary diagnosis of HNSCC. Tumor and normal tissue DNA were genotyped for microsatellite repeat markers in 8 unlinked chromosomal regions. The association between LOH and death from HNSCC was investigated, weighted by number of informative markers per region and adjusted for age at diagnosis, self-reported race, tumor stage and current smoking status. LOH at 3 chromosomal regions were independently associated with reduced survival. A greater risk for cancer mortality was observed for LOH at chromosomal regions 3p24.3-p14.3 (p = 0.02), 8p21.3-p11.21 (p = 0.02) and 9p24.2-p21.2 (p = 0.03). In these regions, LOH at one or more markers was observed in 66.9%, 43.3% and 60.6% of patients, respectively. Survival times were significantly shorter for those with LOH at marker NEFL on 8p21.2 (relative risk = 6.15; p = 0.0002) and at D9S126 on 9p21.2 (relative risk = 5.96; p = 0.0003). Our results indicate that LOH at several chromosomal sites may offer additional independent prognostic information beyond traditional indicators such as tumor stage and age.     

Loss of heterozygosity on chromosome-8 in squamous-cell carcinomas of the head and neck

LOH studies provide evidence for the implication of novel TSGs in human tumours. The p arm of chromosome 8 has been reported to harbour tumour suppressor genes (TSGs) which are very likely to be involved in the development of colon, lung, bladder and hepatocellular carcinomas. In addition, the c-myc proto-oncogene which is located on the 8q arm, has been found to be overexpressed in SCCHN. In the present study we have investigated the incidence of loss of heterozygosity (LOH) in chromosome 8 in 37 tumour specimens of squamous cell carcinomas of the head and neck (SCCHN), using a bank of 11 polymorphic microsatellite markers. The aim of this work was to assess whether there was an 8p TSG(s) in SCCHN, as reported in other tumours and also to investigate whether other areas of chromosome 8 exhibit a high LOH. A relatively high incidence of LOH was found for the markers D8S87 (29%) on 8p12 and ANK1 (20%) on 8p21.2-p11. These two markers are located in the area in which TSG(s) for other cancers have been previously described. When the data on D8S87 and ANK1 were analyzed together it was found that 13/35 (37%) of the SCCHN specimens had a loss at one or other of these markers, thus indicating that a putative TSG(s) in this region may play a role in the development of the SCCHN. No correlation was found between the LOH data and any of the clinicopathological parameters. We also investigated the incidence of c-myc amplification in 144 SCCHN specimens but only 4% were found to have an amplified c-myc allele, thus indicating that the overexpression of c-myc in SCCHN was not the result of gene amplification.     

Evidence for three tumor suppressor loci on chromosome 9p involved in melanoma development

Cytogenetic and loss of heterozygosity (LOH) studies have long indicated the presence of a tumor suppressor gene (TSG) on 9p involved in the development of melanoma. Although LOH at 9p has been reported in approximately 60% of melanoma tumors, only 5-10% of these tumors have been shown to carry CDKN2A mutations, raising the possibility that another TSG involved in melanoma maps to chromosome 9p. To investigate this possibility, a panel of 37 melanomas derived from 35 individuals was analyzed for CDKN2A mutations by single-strand conformation polymorphism analysis and sequencing. The melanoma samples were then typed for 15 markers that map to 9p13-24 to investigate LOH trends in this region. In those tumors demonstrating retention of heterozygosity at markers flanking CDKN2A and LOH on one or both sides of the gene, multiplex microsatellite PCR was performed to rule out homozygous deletion of the region encompassing CDKN2A. CDKN2A mutations were found in tumors from 5 patients [5 (14%) of 35], 4 of which demonstrated LOH across the entire region examined. The remaining tumor with no observed LOH carried two point mutations, one on each allele. Although LOH was identified at one or more markers in 22 (59%) of 37 melanoma tumors corresponding to 20 (57%) of 35 individuals, only 11 tumors from 9 individuals [9 (26%) of 35] demonstrated LOH at D9S942 and D9S1748 the markers closest to CDKN2A. Of the remaining 11 tumors with LOH 9 demonstrated LOH at two or more contiguous markers either centromeric and/or telomeric to CDKN2A while retaining heterozygosity at several markers adjacent to CDKN2A. Multiplex PCR revealed one tumor carried a homozygous deletion extending from D9S1748 to the IFN-alpha locus. In the remaining eight tumors, multiplex PCR demonstrated that the observed heterozygosity was not attributable to homozygous deletion and stromal contamination at D9S1748, D9S942, or D9S974, as measured by comparative amplification strengths, which indicates that retention of heterozygosity with flanking LOH does not always indicate a homozygous deletion. This report supports the conclusions of previous studies that a least two TSGs involved in melanoma development in addition to CDKN2A may reside on chromosome 9p.     

Loss of heterozygosity at chromosomes 3p and 17p in primary non-small cell lung cancer

BACKGROUND: Loss of heterozygosity (LOH) of selected regions at chromosomes 3p and 17p in non-small cell lung cancer (NSCLC) and the association of these abnormalities with major clinical parameters and prognosis were studied. MATERIALS AND METHODS: The study group included 92 consecutive primary NSCLC tumours and four microsatellite markers from chromosome 3p and three markers from 17p were analyzed. RESULTS: LOH of at least one locus was found in 83% of all analyzed tumours. Most frequently deletion (58%) was found at locus D3S1481 (3p14.2). Sequence deletions of D17S520 (17p12) and TP53 (17p13.1) occurred in 52% of tumours. LOH occurrence at 3p and 17p was more frequent in squamous cell carcinomas compared to adenocarcinomas (89% vs. 75%), but this difference was not significant. CONCLUSION: No significant association was found between LOH on any analyzed loci and tumour stage (TNM) and grade (G). There was no correlation between LOH and survival.     

Loss of heterozygosity of 3p markers in neuroblastoma tumours implicate a tumour-suppressor locus distal to the FHIT gene.

Neuroblastoma is a heterogeneous childhood tumour of the sympathetic nervous system, in which deletions of chromosomal region 1p and amplification of the MYCN oncogene correlate with aggressive tumour behaviour. However, the majority of neuroblastoma tumours show neither of these aberrations, indicating that other chromosomal regions may be involved in tumorigenesis. Here, we report findings of loss of heterozygosity (LOH) on chromosome 3. In our neuroblastoma material, nine of 59 (15.3%) tested tumours showed allelic loss of chromosome 3p markers. We found significant clinical and biological differences between tumours with the loss of one entire chromosome 3 vs tumours with partial loss in chromosome region 3p. All children with tumours with whole chromosome 3 loss are long-term survivors, whereas all children with tumours showing partial 3p LOH have died from tumour progression. A consensus region found to be deleted in all the tumours with 3p deletions was defined by markers D3S1286 and D3S1295, i.e. 3p25.3-p14.3, distal to the FHIT gene.     

Refined deletion mapping in sporadic breast cancer at chromosomal region 8p12-p21 and association with clinicopathological parameters

We have further refined the loss of heterozygosity (LOH) pattern on the human chromosomal region 8p12-p21 using 15 well characterised microsatellite markers in a panel of 50 breast carcinomas. The allelic loss pattern of these tumours suggests the presence of five commonly deleted regions on 8p12-p21. The most commonly deleted region was located between markers D8S1734 and D81989, spanning a distance of approximately 3 cM and reaching 56% LOH at locus NEFL. LOH at 8p12-p21 was significantly correlated with large tumour size (T>5 cm). Patients with the age at diagnosis of breast cancer between 45 and 55 years showed significantly more LOH than patients older than 55 years or younger than 45 years. No correlation was observed between 8p12-p21 alterations and histological tumour type, grade and the presence of lymph node metastases.     

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.     

Frequent 3p allele loss and epigenetic inactivation of the RASSF1A tumour suppressor gene from region 3p21.3 in head and neck squamous cell carcinoma

Studies of allelic imbalance and suppression of tumourigenicity have consistently suggested that the short arm of chromosome three (3p) harbours tumour suppressor genes (TSGs) whose inactivation leads to the development of various types of neoplasia including head and neck squamous cell carcinoma (HNSCC). Previously, we defined a critical minimal region of 120kb at 3p21.3 that contains overlapping homozygous deletions in lung and breast tumour lines and isolated eight genes from the minimal region. Mutation analysis in a large panel of lung and breast cancers revealed only rare mutations, but the majority of lung tumour lines showed loss of expression for one of the eight genes (RASSF1A) due to hypermethylation of a CpG island in the promoter region of RASSF1A. We found RASSF1A to be methylated in the majority of lung tumours, but to a lesser extent in breast and ovarian tumours. In order to define the role of 3p TSGs, in particular RASSF1A in HNSCC, we (a) analysed 43 primary HNSCC for allelic loss in regions proposed to contain 3p TSGs (3p25-26, 3p24, 3p21-22, 3p14 and 3p12), (b) analysed 24 HNSCC for evidence of RASSF1A methylation and (c) undertook mutation analysis of RASSF1A in HNSCC. We found that 81% of HNSCC showed allele loss at one or more 3p markers, 66% demonstrated loss for 3p21.3 markers and 56% showed allelic losses at 3p12 loci. Thus, 3p loss is common in HNSCC and extensive 3p loss occurs even in early stage tumours. RASSF1A promoter region hypermethylation was found in 17% (4/24) of the sporadic HNSCC, but RASSF1A mutations were not identified. Furthermore, we found RASSF1A methylation to be significantly higher in poorly differentiated then in moderate to well differentiated HNSCC (P=0.0048). Three of the four tumours showing RASSF1A methylation also underwent 3p21.3 allelic loss, hence RASSF1A behaves as a classical TSG (two hits, methylation and loss). One tumour with RASSF1A methylation had retention of markers at 3p providing further evidence of specific inactivation of RASSF1A as a critical step in some HNSCC. Although the frequency of 3p21.3 allele loss was substantially higher than that of RASSF1A methylation this does not necessarily suggest that other genes from 3p21.3 are also implicated in HNSCC, as 3p21.3 LOH was invariably found with LOH at other 3p loci. Thus, the presence of 3p21.3 allele loss without RASSF1A methylation might reflect a propensity for 3p21.3 loss to occur as a secondary consequence of large 3p deletions targeted at other 3p TSG regions. Furthermore, in the presence of homozygous inactivation of other 3p TSGs, RASSF1A haploinsufficiency might be sufficient to promote tumourigenesis in many HNSCC.     

High resolution mapping of chromosome 6 deletions in cervical cancer.

Chromosome 6 is frequently affected in different tumors. However, little information exists on chromosome 6 deletions in cervical cancer. We have studied loss of heterozygosity (LOH) and microsatellite instability (MIN) in 62 invasive squamous cell carcinomas of the cervix (CC) using 19 polymorphic microsatellite markers spanning both arms of chromosome 6 and one marker located at 5p15. We found that LOH at chromosome 6 is a common feature of cervical carcinomas: 90% (56/62) of CC had LOH at least at one locus and about 58% (36/62) had LOH on both arms of chromosome 6. The highest LOH incidence was shown in HLA region (6p21.3-6p21.1) with markers D6S273 and D6S276 in 52.7% and 45.2% of informative cases respectively. Frequent LOH on 6q was found at loci D6S311 (6q24-25. 1), D6S305 (6q26) and D6S281 (6q27-6qter) in 37.8%, 33.3% and 39.0% of informative cases respectively. There was no significant correlation observed between clinical parameters of cervical cancer (age, histologic grade, clinical stages and progression) and LOH frequency. Microsatellite instability was found in 3 out of 62 cases (4.8%) at three and more loci out of 20 tested. The study shows that several regions on 6p and 6q may harbour potential tumor-suppressor genes important for cervical cancer progression.     

Detailed genetic and physical mapping of tumor suppressor loci on chromosome 3p in ovarian cancer.

Hemizygosity and homozygosity mapping studies show that many common sporadic cancers including lung, breast, kidney, cervical, ovarian, and head and neck cancer display deletions on the short arm of chromosome 3. For ovarian cancer, monochromosomal transfer suppression studies have identified three candidate regions for chromosome 3p ovarian cancer tumor suppressor genes (OCTSGs). To accurately map OCTSG candidate regions, we analyzed 70 ovarian tumors for loss of heterozygosity (LOH) at 20 loci on chromosome 3p that were selected to target those regions proposed to contain tumor suppressor genes for common sporadic cancers. All samples were informative for at least five markers. In 33 (52%) tumors without microsatellite instability, LOH was observed for at least one 3p marker. Analysis of 27 ovarian tumors demonstrating both loss and retention of 3p markers enabled us to define four nonoverlapping minimal deletion regions (OCLOHRs): (a) OCLOHR-1 mapped distal to D3S3591 at 3p25-26; (b) OCLOHR-2 mapped between D3S1317 and D3S1259 at 3p24-25; (c) OCLOHR-3 mapped between D3S1300 and D3S1284, an area that includes the FHIT locus at 3p14.2; and (d) OCLOHR-4 mapped between D3S1284 and D3S1274 at 3p12-13, a region known to contain overlapping homozygous deletions in lung and breast tumor cell lines. However, microsatellite markers from the chromosome 3p21.3 interval homozygously deleted in lung cancer cell lines did not identify a distinct OCLOHR. The frequency and extent of 3p LOH correlated with tumor stage such that LOH at two or more OCLOHRs was present in 53% (16 of 30) of stage III tumors but only 26% (5 of 19) of stage I/II tumors (P = 0.08). To determine the relationship between the OCLOHRs and the three candidate ovarian cancer suppression regions (OCSRs) identified previously by monochromosome transfer studies, we performed detailed genetic and physical mapping studies to define the extent of the three candidate OCSRs and to establish YAC contigs covering each region. OCSR-A at 3p25-26 and OCSR-B at 3p24 were shown to overlap with OCLOHR-1 and OCLOHR-2, respectively, providing further evidence for OCTSGs in these regions. We also show that OCSR-C overlaps with a locus at 3p21.3 previously implicated in lung and breast cancer.