Microsatellite analysis at 1p36.3 in malignant melanoma of the skin: fine mapping in search of a possible tumour suppressor gene region

Deletions in 1p36 in malignant melanoma have been found in high percentages in nodular melanomas and melanoma metastases. Despite many efforts, no candidate tumour suppressor gene associated with malignant melanoma has so far been found in this region. To further determine a possible tumour suppressor gene locus, we carried out a deletion mapping of chromosome 1p36 at nine microsatellite loci in 74 malignant melanomas. Loss of heterozygosity (LOH) in this region was found in 77% of nodular melanomas (NMs), 86% of metastatic melanomas, but only 20% of superficial spreading melanomas (SSMs). Regarding the allelic losses, the nodular and metastatic melanoma samples could be divided into three groups: one showing LOH at the more telomeric loci D1S243 and D1S468 (1p36.33), one displaying allelic loss at the more centromeric loci D1S214 and D1S253 (1p36.32-31) and one with LOH over all informative loci between D1S243 and D1S160. We did not find any significant correlation between a deletion in any of the investigated loci and the survival data of the patients. However, our results confine the deleted region in malignant melanoma to a very small area around 1p36.32, thus facilitating the search for the tumour suppressor gene with importance in malignant melanoma.     

The genomic structure of the DMBT1 gene: evidence for a region with susceptibility to genomic instability

Increasing evidence has accumulated for an involvement of the inactivation of tumour suppressor genes at chromosome 10q in the carcinogenesis of brain tumours, melanomas, and carcinomas of the lung, the prostate, the pancreas, and the endometrium. The gene DMBT1 (Deleted in Malignant Brain Tumours 1) is located at chromosome 10q25.3-q26.1, within one of the putative intervals for tumour suppressor genes. DMBT1 is a member of the scavenger-receptor cysteine-rich (SRCR) superfamily and displays homozygous deletions or lack of expression in glioblastoma multiforme, medulloblastoma, and in gastrointestinal and lung cancers. Based on these properties, DMBT1 has been proposed to be a candidate tumour suppressor gene. We have determined the genomic sequence of DMBT1 to allow analyses of mutations. The gene has at least 54 exons that span a genomic region of about 80 kb. We have identified a putative exon with coding potential for a transmembrane domain. Our data further suggest that alternative splicing gives rise to isoforms of DMBT1 with a differential utilization of SRCR domains and SRCR interspersed domains. The major part of the gene harbours locus specific repeats. These repeats may point to the DMBT1 locus as a region susceptible to chromosomal instability.     

Simple tandem repeat allelic deletions confirm the preferential loss of distal chromosome 6q in melanoma

Karyotypic analysis, loss of somatic heterozygosity, microcell fusion and cDNA transfection studies have provided compelling evidence that at least one tumour suppressor gene for melanoma resides on chromosome 6. In an attempt to further define the regions to which these putative suppressor genes map, we have carried out loss of heterozygosity (LOH) studies on DNA from 25 fresh melanoma tumours for 9 simple tandem repeat (STR) polymorphism markers spanning chromosome 6. Four samples displayed LOH or homozygosity for all markers studied, indicating that they had lost one homologue of chromosome 6. An additional 3 samples showed LOH for all markers on 6q. Furthermore, 30 melanoma cell lines, for which there were no matching somatic DNA samples, were analyzed for hemizygosity of markers on 6q. One cell line had a homozygous deletion of all markers tested and a further 12 cell lines displayed only one allele for 3 or 4 contiguous markers, indicating that most, if not all of these samples were hemizygous for the region of 6q distal to D6S87. Overall, the rate of LOH on 6q in the 55 melanoma DNAs was 35%, and there were no losses of markers on 6p without concomitant loss of markers on 6q. Two of 5 samples derived from primary melanomas showed LOH, which indicates that LOH for the melanoma suppressor gene on 6q, which maps to a region that contains the SOD2 locus, is a frequent and early event in melanoma tumorigenesis.     

PTEN inactivation is rare in melanoma tumours but occurs frequently in melanoma cell lines

Deletions detected in cytogenetic and loss of heterozygosity (LOH) studies indicate that at least one tumour suppressor gene maps to the long arm of chromosome 10. Previous deletion mapping studies have observed LOH on 10q in about 30% of melanomas analysed. The PTEN gene, mapping to chromosome band 10q23.3, encodes a protein with both lipid and protein phosphatase activity. Somatic mutations and deletions in have been detected in a variety of cell lines and tumours, including melanoma samples. We performed mutation analyses and extensive allelic loss studies to investigate the role this gene plays in melanoma pathogenesis. We found that a total of 34 out of 57 (60%) melanoma cell lines carried hemizygous deletions of chromosome 10q encompassing the PTEN locus. A further three cell lines carried smaller deletions excluding PTEN. Inactivation of both PTEN alleles by exon-specific homozygous deletion or mutation was observed in 13 out of 57 (23%) melanoma cell lines. The mutation spectrum observed does not indicate an important role for ultraviolet radiation in the genesis of these mutations, and evidence from three cell lines supports the acquisition of PTEN aberrations in culture. Ten out of 49 (20%) matched melanoma tumour/normal samples harboured hemizygous deletions of either the whole chromosome or most of the long arm. Mutations within were detected in only one of the 10 tumours demonstrating LOH at 10q23 that were analysed. These results suggest that PTEN inactivation may be important for the propagation of melanoma cells in culture, and that another chromosome 10 tumour suppressor gene may be important for melanoma pathogenesis.     

Melanoma ex naevo: a study of the associated naevus

It has been shown that the co-occurrence of melanoma and pre-existing naevus is not a random event and that acquired naevi may be precursors of melanoma. A critical area of chromosomal loss at 9p21 has been implicated in the genesis of malignant melanoma, representing a site of frequent somatic chromosomal deletions in melanoma. Allelic deletions within this chromosomal region most often include the tumour suppressor gene p16. The objective of this study was to search for allelic deletions on chromosome 9p21 in naevus cell clusters. A microdissection-based approach was used to analyse 30 archived primary cutaneous melanomas and associated naevi for loss of heterozygosity (LOH) at 9p21 using the polymorphic DNA markers D9S171 and IFNA. LOH was detected in 10 out of 27 informative naevi (37%) at D9S171 and in eight out of 19 (42%) at IFNA in the dissected naevus cell clusters, and in nine out of 27 (33%) at D9S171 and seven out of 19 (36%) at IFNA in the associated melanomas. In eight out of 46 (17%) cases, LOH was detected simultaneously in the naevus and the associated melanoma using both markers. Our results suggest a causal relationship for the development of melanoma within a pre-existent associated naevus. These data support the hypothesis that lesions within 9p21 play an important role in early melanoma development, since these genetic alterations are found in histologically benign melanoma-associated naevi.     

Frequent loss of heterozygosity on chromosome 6 in human ovarian carcinoma.

Investigation of genetic changes in tumours by loss of heterozygosity (LOH) is a powerful technique for identifying chromosomal regions that may contain tumour suppressor genes. LOH has been described on chromosome 6 in ovarian carcinoma using restriction fragment length polymorphism analysis with a small number of probes. We studied 29 ovarian carcinomas with 19 probes mapping to chromosome 6. Sixteen of the 29 tumours showed LOH on 6q (55%). Of these 16, 63% showed loss of all informative markers on that arm. One tumour showed loss of 6q24-qter, localising the putative tumour suppressor gene to that region. Loss on 6p was 28% overall. However, using three dinucleotide repeat primer pairs from 6p to study LOH in seven selected tumours, LOH was demonstrated at both 6p22.3-pter and at 6p12-6p22. These results confirm that 6q harbours a tumour suppressor gene of relevance to ovarian carcinoma and suggest that there may also be a similar gene(s) on 6p. By Southern analysis, there was no evidence of genomic rearrangements of the oestrogen receptor gene, located at 6q25.1. LOH on 6q was more common in high than low grade tumours. The relevance of our findings to previous work in ovarian cancer and other solid tumours is discussed.     

Destabilization of chromosome 9 in transitional cell carcinoma of the urinary bladder.

The most frequent genetic alteration in transitional cell carcinoma of the urinary bladder (TCC) is loss of chromosome 9 which targets CDKN2A on 9p. The targets on 9q are not confirmed. Here, 81 advanced TCC specimens were investigated for loss of heterozygosity (LOH) and homozygous deletions (HD) on chromosome 9q using multiplex analysis of microsatellite markers. 41/81 tumours (51%) showed LOH on 9q, with LOH at all markers in 33 cases. Eight partial losses involved three regions in 9q12, 9q22.3, and 9q33- 9q34. No mutations were identified in the candidate tumour suppressor gene DBCCR1 in three tumours showing restricted LOH at 9q32-33. 22% of the specimens had HD at CDKN2A, but no HD was found on 9q. Two tumours had lost 9p only and five 9q only. 9q LOH was not related to tumour grade or stage and present or absent with equal frequency in recurrent TCC. LOH on 9q correlated with the extent of genome-wide hypomethylation (P < 0.0001) which extended into satellite sequences located in 9q12 juxtacentromeric heterochromatin. While the high frequency of chromosome 9q loss in TCC may reflect destabilization of the chromosome related to hypomethylation of repetitive DNA, the data are compatible with the existence of tumour suppressor genes on this chromosome arm.     

Concurrent LOH at multiple loci in human malignant mesothelioma with preferential loss of NF2 gene region

Human malignant mesothelioma (MM) is a highly aggressive neoplasm related to occupational asbestos exposure and characterised by a long latency period between the exposure and onset of disease. Previous studies indicate that losses at different genomic regions are present in MM. We examined allele loss at three known tumour suppressor gene regions (22q/NF2 gene, 9p/p16 gene, and 3p/FHIT gene) and at two other frequently deleted areas (14q and 6q) in MM. Loss of heterozygosity (LOH) was investigated in cell cultures and primary tumours with several highly polymorphic markers for each site. To study if LOH of the NF2 gene is a consistent feature in MM, we performed a more detailed analysis of chromosome 22q that included a NF2 marker (NF2CA3). We observed a high frequency of LOH occurring simultaneously at multiple loci. In particular, 100% of the cultured MM cells exhibited LOH at the NF2 gene region. From the other chromosomal sites analysed, recurrent allele loss was detected at 9p (5/7; 71%), 3p (4/7; 57%), 14q (3/7; 43%), and 6q (3/7; 43%). Of the 32 tumours, even those trimmed to exclude normal tissue, few showed LOH, suggesting consielment by normal cells within MM tumours, whereas tumour cells in primary cultures showed LOH already in passages 1-2. In conclusion, our present LOH data indicate that MM cells exhibit allele losses at multiple tumour suppressor gene sites concurrently, involving NF2 gene preferentially. This supports the view that the accumulation of multiple genetic hits is characteristic to malignant transformation of MM cells.     

Somatic mutation of PTEN in bladder carcinoma.

The tumour suppressor gene PTEN/MMAC1, which is mutated or homozygously deleted in glioma, breast and prostate cancer, is mapped to a region of 10q which shows loss of heterozygosity (LOH) in bladder cancer. We screened 123 bladder tumours for LOH in the region of PTEN. In 53 informative muscle invasive tumours (> or = pT2), allele loss was detected in 13 (24.5%) and allelic imbalance in four tumours (overall frequency 32%). LOH was found in four of 60 (6.6%) informative, non-invasive tumours (pTa/pT1). We screened 63 muscle invasive tumours for PTEN mutations by single-strand conformation polymorphism (SSCP) analysis and for homozygous deletion by duplex quantitative polymerase chain reaction (PCR). Two homozygous deletions were identified but no mutations. Of 15 bladder tumour cell lines analysed, three showed homozygous deletion of all or part of the PTEN gene, but none had mutations detectable by SSCP analysis. Our results indicate that PTEN is involved in the development of some bladder tumours. The low frequency of mutation of the retained allele in tumours with 10q23 LOH suggests that there may be another predominant mechanism of inactivation of the second allele, for example small intragenic deletions, that hemizygosity may be sufficient for phenotypic effect, or that there is another target gene at 10q23.     

DMBT1 is frequently downregulated in well-differentiated gastric carcinoma but more frequently upregulated across various gastric cancer types

Well-differentiated gastric carcinomas are considered to represent a distinct entity emerging via specific molecular changes different from those found in other gastric carcinoma types. The gene deleted in malignant brain tumours 1 (DMBT1) at 10q25.3-q26.1 codes for a protein presumably involved in cell differentiation and protection and has been proposed as a candidate tumour suppressor for brain and epithelial cancer. One study reported a loss of DMBT1 expression in 12.5% (5/40) of gastric cancer samples. Here, we examined in more detail DMBT1 protein and mRNA expression in 78 primary gastric tumour samples and corresponding normal gastric mucosa. DMBT1 was expressed in all non-tumour gastric mucosa tissues. Eleven out of 71 (15%) gastric tumours were negative for the DMBT1 protein in immunohistochemical analyses. Lack of DMBT1 expression was significantly more frequently found in well-differentiated gastric tumours (6/18 well-differentiated tumours vs. 5/53 other subtypes; P=0.025). Quantitative RT-PCR revealed a downregulation of the DMBT1 mRNA for 8/21 (38%) cases, while the remaining 13 cases (62%) displayed a substantial upregulation. Our data suggest that a loss of DMBT1 expression may preferentially take place in well-differentiated gastric carcinoma. However, an upregulation of DMBT1 expression is more frequently found across all gastric cancer types.     

Frameshift mutations of RIZ,but no point mutations in RIZ1 exons in malignant melanomas with deletions in 1p36

Recently, the retinoblastoma protein interacting zinc finger gene RIZ has been proposed as a candidate for the tumor suppressor locus on 1p36, because of the common loss of RIZ1 RNA in human tumors. In addition, frameshift mutations of this gene have been demonstrated in a variety of tumors with microsatellite instability. Since alterations in this region have been described in malignant melanomas, we investigated DNA of paraffin-embedded sections from 16 typical naevi, 19 atypical naevi, 33 primary melanoma lesions and 25 metastases and DNA from four melanoma cell lines by PCR and direct sequencing analysis of RIZ. Frameshift mutations in the RIZ gene were found in 17% of melanoma samples and 8.6% of naevi, but we could not demonstrate any missense mutations in the exons of RIZ1. No LOH of the RIZ gene nor any microsatellite instability in six dinucleotide markers or in the mononucleotide repeats IGRIIR, hMSH3, and hMSH6 could be demonstrated in the samples with RIZ frameshift mutations. Although our results do not explain the high rate of deletions in 1p36 found in this tumor, they assign RIZ a potential role in the multi-step tumor forming process of malignant melanoma of the skin.     

Expression of the DMBT1 Gene Is Frequently Suppressed in Human Lung Cancer

DMBT1 (deleted in malignant brain tumors) is a candidate tumor suppressor gene that has been mapped to chromosome 10q25.3-q26.1, a region in which frequent loss of heterozygosity (LOH) has been observed in several human tumors. Since DMBT1 is highly expressed in the lung, we analyzed LOH at the DMBT1 locus and expression of this gene in lung cancer. Thirty-five (53%) of 66 primary lung cancers showed LOH, and diminished expression of DMBT1 was observed in 20 (91%) of 22 lung cancer cell lines: three (14%) of them showed loss of expression. We further determined the primary structure of DMBT1 and analyzed genetic alterations in this gene using 23 lung cancer cell lines. Two (9%) of them had homozygous deletion within the gene, and two cell lines had genetic aberrations: one was a rearrangement involving exons 5 and 6, and the other was a missense mutation at codon 52. These results suggest that inactivation of the DMBT1 gene plays an important role in human lung carcinogenesis.     

Expression of the DMBT1 gene is frequently suppressed in human lung cancer.

DMBT1 (deleted in malignant brain tumors) is a candidate tumor suppressor gene that has been mapped to chromosome 10q25.3-q26.1, a region in which frequent loss of heterozygosity (LOH) has been observed in several human tumors. Since DMBT1 is highly expressed in the lung, we analyzed LOH at the DMBT1 locus and expression of this gene in lung cancer. Thirty-five (53%) of 66 primary lung cancers showed LOH, and diminished expression of DMBT1 was observed in 20 (91%) of 22 lung cancer cell lines: three (14%) of them showed loss of expression. We further determined the primary structure of DMBT1 and analyzed genetic alterations in this gene using 23 lung cancer cell lines. Two (9%) of them had homozygous deletion within the gene, and two cell lines had genetic aberrations: one was a rearrangement involving exons 5 and 6, and the other was a missense mutation at codon 52. These results suggest that inactivation of the DMBT1 gene plays an important role in human lung carcinogenesis.     

Loss of expression of the metastasis suppressor gene KiSS1 during melanoma progression and its association with LOH of chromosome 6q16.3-q23

KiSS1 is a putative melanoma metastasis suppressor gene, the expression of which may be regulated by another gene(s) mapping to chromosome 6q16.3-q23. To additionally elucidate the role of KiSS1 in the progression of human melanoma in vivo, we examined KiSS1 mRNA expression in 51 melanocytic tumors with various stages of progression by in situ hybridization. We also examined a correlation between loss of KiSS1 mRNA expression and loss of heterozygosity (LOH) of 6q16.3-q23 in 27 melanoma metastases. All of the four nevocellular nevi and eight primary melanomas <4 mm in thickness showed KiSS1 mRNA expression, whereas only 50% (6 of 12) of primary melanomas >4 mm in thickness expressed KiSS1. Loss of KiSS1 mRNA was equally frequent in metastases; 44% (12 of 27) of tumors lost KiSS1 expression. LOH of 6q16.3-q23 was observed in 52% (14 of 27) of metastases. There was a strong association between LOH and loss of KiSS1 expression (P = 0.03); nine metastases with LOH of 6q16.3-q23 lost KiSS1 expression, whereas 10 tumors with no LOH showed positive KiSS1 mRNA expression. The findings in this study show, for the first time, KiSS1 down-regulation during the progression of melanoma in vivo and strongly suggest that inactivation of a tumor suppressor gene(s) mapping to 6q16.3-q23 by deletion or mutation coupled with LOH may lead to the down-regulation of KiSS1.     

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.     

Identification of distinct regions of allelic loss on chromosome 13q in nasopharyngeal cancer from paraffin embedded tissues.

Our main purpose was to identify tumor suppressor gene loci on chromosome 13 responsible for nasopharyngeal cancer (NPC) development by analyzing loss of heterozygosity (LOH) and RB protein expression in paraffin embedded tissues. Normal and tumor DNA were extracted from microdissected samples, and their whole genomes were amplified using degenerate oligonucleotide primers. The polymerase chain reaction (PCR) products were analyzed by repeated amplification using primers derived from 16 microsatellite regions spanning the long arm of this chromosome. Among 50 informative cases, LOH was observed in 44 tumors. Thirty-one tumors displayed partial loss and provided an informative basis for detailed deletion mapping. Three minimal regions of loss were delineated; the first flanked by D13S120 and D13S219, the second by D13S126 and D13S119, and the third by D13S137 and 13qter. These 3 regions were linked to BRCA2 on 13q12, RB1 on 13q14, and 13q14.3-ter, respectively. Seven and 4 cases showed LOH either on 13q12 or 13q14, respectively. Nineteen cases showed LOH of both loci separately. One NPC displayed 13q12 and 13q14.3-ter LOH. RB protein expression was detectable in 76% of the cases. Ten out of 15 cases with the allelic losses limited to 13q14 showed RB protein expression. Contrasting that, 6 out of 7 cases devoid of RB protein expressions showed 13q14LOH. In conclusion, 13qLOH, involving 3 tumor suppressor gene loci, appears to be a frequent genetic event occurring during NPC development. However, other tumor suppressor genes besides RB1, may be responsible for the majority of 13q14LOH.     

Loss of heterozygosity at 9q33 and hypermethylation of the DBCCR1 gene in oral squamous cell carcinoma

The DBCCR1 gene at chromosome 9q33 has been identified as a candidate tumour suppressor, which is frequently targeted by promoter hypermethylation in bladder cancer. Here, we studied the possible involvement of DBCCR1 in the development of oral squamous cell carcinoma. DNA from 34 tumours was examined for loss of heterozygosity (LOH) at three markers surrounding DBCCR1 and for hypermethylation of the DBCCR1 promoter, using methylation-specific PCR and methylation-specific melting-curve analysis. LOH was found in 10 of 31 cases (32%), and DBCCR1 hypermethylation was present in 15 of 34 cases (44%). Hypermethylation of DBCCR1 was also present in three of seven epithelial tissues adjacent to the tumours, including two hyperplastic and one histologically normal epithelia. Furthermore, of four oral leukoplakias with dysplasia, one showed LOH at 9q33 and two showed DBCCR1 hypermethylation. These data suggest that LOH at 9q33 and hypermethylation of the DBCCR1 promoter are frequent and possibly early events in oral malignant development.