Overexpression and mutations of p53 in metastatic malignant melanomas

Alterations of the p53 tumor suppressor gene are the most frequent genetic abnormalities in human malignancies, but the role of p53 in the etiology of malignant melanomas is unclear. Fifty unselected malignant melanomas were analyzed for p53 overexpression by immunohistochemistry using 3 monoclonal antibodies (MAbs). Fifteen tumors (29.4%) showed positive staining with at least 2 different antibodies. In the first 20 consecutive tumors exons 5–9 and adjacent splice sites of the p53 gene were analyzed by genomic sequencing. There were 4 mutations in 20 metastatic melanomas. Three of 4 mutations were C:G → T:A transitions. A search of our database of p53 mutations revealed that out of 8 p53 mutations reported by others, 4 are C:G → T:A transitions at dipyrimidine sites, and one is a tandem CC → TT mutation. This mutational pattern is comparable with the pattern of p53 mutations in squamous cell and basal cell carcinomas of the skin and is related to exposure to ultraviolet B (UV-B) wavelength radiation. Taken together with a predominance of UV-induced mutations in the CDKN2/p16 gene demonstrated in melanoma cell lines, our data support a role of sunlight exposure in the etiology of malignant melanoma. The low frequency of p53 mutants in melanomas compared with other types of skin cancers suggests that although mutations in this gene are likely to be involved in the development of some malignant melanomas, they do not play as large a role as in squamous and basal cell carcinomas of the skin. © 1996 Wiley-Liss, Inc.     

Homozygous deletions but no sequence mutations in coding regions of p15 or p16 in human primary bladder tumors

Chromosome 9p21 appears to harbor a tumor suppressor gene, as evidenced by deletions in this region in a variety of human primary tumors and cell lines. To map the deletion at 9p21 in bladder tumors, we analyzed DNA from 28 tumor and normal pairs at five microsatellite markers that flank the region occupied by the putative tumor suppressor genes p16 and p15. Loss of heterozygosity (a) at the markers human interferon (a) α and D9S171, which are adjacent to the p15 and p16 loci, was detected in 41% and 33%, respectively, of informative cases of bladder tumors. No sequence mutations were detected in exons 1 or 2 of either p15 or p16 in any of the bladder tumors. Three sequence-tagged site markers in the region bordered by HIFNα and D9S171 were used to further map the deleted region by multiplex polymerase chain reaction with the HIFNγ marker (on chromosome 12) as a control for amplification. Six of 11 tumors with LOH at surrounding markers had homozygous deletions of the marker c5.1, which is located within the p16 gene; and two tumors appeared to have homozygous deletions within p15(RN1.1) but not p16 (c5.1). A recently identified microsatellite marker, p16-CA-1, located 16 kb distal to p16, proved valuable in defining the minimal deletion involved in these bladder tumors. Five tumors exhibited homozygous deletions of this marker but not HIFNα and two tumors showed LOH at this marker and homozygous deletion of p16. Although these data could not be used to identify p16 or p15 as the definitive tumor suppressor gene in this region that is involved in bladder carcinogenesis, they suggest that homozygous deletion is a common mechanism of loss of tumor suppressor gene function in this region. © 1995 Wiley- Liss, Inc.     

AXIN1 mutations but not deletions in cerebellar medulloblastomas

Medulloblastoma is a malignant, invasive embryonal tumour of the cerebellum which manifests preferentially in children. A subset of cases is associated with colon cancer and APC germline mutations (Turcot syndrome), and APC and beta-catenin point mutations occur in up to 10% of sporadic cases, indicating the involvement of the Wnt pathway in the development of medulloblastoma. In 39 sporadic cerebellar medulloblastomas screeened for alterations in the AXIN1 gene, another component of the Wnt pathway, we found missense AXIN1 mutations in two tumours, CCC-->TCC at codon 255 (exon 1, Pro-->Ser) and TCT-->TGT at codon 263 (exon 1, Ser-->Cys). Furthermore, the A allele at the G/A polymorphism at nucleotide 16 in intron 4 was significantly over-represented in medulloblastomas (39 cases; G 0.76 vs-A 0.24) compared to healthy individuals (86 cases; G 0.91 vs A 0.09; P=0.0027). RT-PCR revealed large deletions in the AXIN1 gene in 5/12 (42%) medulloblastomas, consistent with a previous report. However, we observed such deletions at a similar frequency also in normal brain tissue (6/12, 50%). Since there are multiple complementary, inverted sequences present in the AXIN1 gene, these large deletions may represent RT-PCR errors due to stem-loop secondary structures.     

Aberrant Methylation of the 1p36 Tumor Suppressor Gene RIZ1 in Renal Cell Carcinoma

Background: Retinoblastoma protein-interacting zinc finger gene 1(RIZ1) functions as a tumor suppressor.Hypermethylation-mediated RIZ1 silencing has been reported in several cancers, but not in renal cell carcinoma(RCC) yet. Materials and Methods: We examined the RIZ1 expression and methylation in a panel of RCC celllines and 50 primary tumors using semiquantitative/quantitative polymerase chain reaction (PCR), methylationspecific PCR, and bisulfite sequencing genomic. We also explored the relationship between methylation status ofRIZ1 and clinicopathological features in RCC patients. Results: RIZ1 expression was down-regulated or lost inOS-RC-2, 769-P, Caki-1, 786-O and A498 RCC cell lines. Restored expression of RIZ1 was detected after additionof 5-aza-2’-deoxycytidine with/without trichostatin A, suggesting that DNA methylation directly mediates itssilencing. The RIZ1 expression was significantly reduced in RCCs compared to adjacent non-malignant renalsamples (P<0.001). Aberrant methylation was detected in 15 of 50 (30%) RCCs and in 2 of 28 (7%) adjacent nonmalignantrenal samples (P=0.02). No statistically significant correlation between methylated and unmethylatedcases with regard to age, gender, pathological stage and grade was observed. Conclusions: RIZ1 expression isdown-regulated in human RCC, and this down-regulation is associated with methylation. RIZ1 methylation mayplay a role in renal carcinogenesis.     

Deletions and altered expression of the RIZ1 tumour suppressor gene in 1p36 in pheochromocytomas and abdominal paragangliomas

Pheochromocytomas and abdominal paragangliomas are rare catecholamine-producing tumours arising from neural crest-derived chromaffin cells. Frequent deletions of several distinct regions on the short arm of chromosome 1 suggest their involvement in the tumourigenesis process. The RIZ1 tumour suppressor encoded by the RIZ gene in 1p36.21 represents an attractive candidate target for the distal 1p deletions in these tumours. A panel of 18 pheochromocytomas (14 benign, and 4 malignant) and 11 abdominal paragangliomas (4 benign, and 7 malignant) were characterised for somatic deletions and mRNA expression status of RIZ1 using loss of heterozygosity (LOH) analysis and real-time quantitative PCR, respectively. Furthermore, we evaluated the methylation status of the RIZ1 promoter utilising methylation-specific PCR (MSP). Intragenic LOH at the RIZ locus was detected in 10 of 16 informative cases (62%), including 8 of 12 pheochromocytomas (67%) and 2 of 4 paragangliomas (50%). RIZ1 mRNA appeared to be significantly under-expressed in the tumour samples compared to normal adrenal controls (mean 0.6 vs. 1.0, p<0.001). This was not associated with RIZ1 promoter methylation in any of the samples, indicating that promoter hypermethylation is unlikely to be the underlying cause of the frequent expressional silencing. The recurrent inactivation of the tumour suppressor RIZ1 suggests that this event may be a significant contributing factor to tumour development in pheochromocytomas and abdominal paragangliomas.     

Chromosome 1p and 19q deletions in malignant glioneuronal tumors with oligodendroglioma-like component

Malignant glioneuronal tumors (MGNT) are suggested to be a new entity of glioma defined morphologically as any malignant glioma showing immunohistoichemical evidence of neuronal differentiation. We encountered seven cases of MGNT with oligodendroglioma-like component and investigated alternations of chromosome 1p and 19q in these tumors. Seven patients ranged from 33 to 62 years of age, four females and three males. Immunohistochemical study of these tumors was performed using neuronal markers (synaptophysin, neurofilament, β-tubulin, chromogranin A and NeuN), astrocytic marker (GFAP) and Ki-67. We undertook a molecular cytogenetic study of tumor specimens obtained from seven patients using fluorescence in situ hybridization (FISH) with DNA probes mapping to chromosome 1p36, 1q25, 19p13 and 19q13. Histologically, these tumors resembled anaplastic oligodendroglioma. Immunohistochemically, tumor cells were immunoreactive for synaptophysin (7/7), neurofilament (6/7), β-tubulin (5/7), chromogranin A (4/7), NeuN (2/7) and GFAP (7/7). The Ki-67 labeling index ranged from 4.5% to 20.7%. FISH analysis demonstrated either 1p or 19q deletion in all seven cases (100%) and both 1p and 19q deletions in five cases (71%). The 1p deletion was detected in six of seven cases (86%) and 19q deletion was also detected in six (86%). 1p and 19q deletions were present in MGNT, especially those with oligodendroglial components. We suggest that the oligodendroglial-like feature was associated with not only 1p or 19q loss but also differentiation along neuronal cell lines as a factor of favorable prognosis in glial tumors. It is inappropriate to make a diagnosis of oligodendroglioma based only on morphological resemblance to oligodendroglia.     

Chromosome 1p36 and 22qter deletions in paraffin block sections of intracranial meningiomas

Meningiomas are the most frequent benign tumors of the intracranial cavity. The classification and underlying pathogenetic mechanisms have been reported to be investigated by both pathological and genetic methods. In this study, we aimed to detect Ip36 and 22qter deletions by fluorescence in situ hybridization (FISH) in archival materials of 50 intracranial meningioma patients. The clinical material consisted of paraffin-embedded tissue sections from 50 patients who were surgically treated and had histopathologic diagnosis of an intracranial meningioma. We observed Ip36 deletion in 23/50 (46%) and 22qter deletion in 33/50 (66%) patients. In addition, we observed 22qter deletion in 26/36 (72.2%) patients with meningothelial meningioma. This finding implies that 22qter deletion might play an important role in the pathogenesis of meningothelial meningioma. On the other hand, no alterations were documented in the frequency of these chromosomal alterations according to the grade of meningiomas, suggesting that malignant progression of these tumors depends on other, more relevant, genetic changes.     

p53 protein expression and TP53 mutations in malignant melanomas of sun-sheltered mucosal membranes versus chronically sun-exposed skin

In this paper, we compare the expression of the TP53 gene product, p53 protein (p53p), in primary malignant melanomas from sun-shielded mucous membranes and from chronically sun-exposed skin. Archival tissues from 29 patients with mucosal melanomas and from 27 with cutaneous melanomas in facial skin were subjected to immunohistochemical procedures using the monoclonal antibody DO-1. p53p expression did not differ significantly between the two groups of melanomas. A comparison with previously obtained data on TP53 mutations from the same tumours showed closer concordance amongst mucosal than amongst skin tumours. Primary mucosal melanomas and their satellites showed identical patterns, focal or diffuse, of p53p expression. Thus, expression of altered p53p could well participate in the clonal expansion of these mucosal melanomas and in tumour progression. The p53p characteristics recognized in our investigations are amongst the first hallmarks in the emerging molecular pathological profiling of mucosal melanomas, and may therefore be useful in exploring the aetiology of UV-independent melanomas.     

Multiple polymorphisms, but no mutations, in the WAF1/CIP1 gene in human brain tumours.

The cyclin kinase inhibitor WAF1/CIP1, also termed CDKN1, mediates p53-induced cell cycle arrest in response to DNA damage. This property makes it an attractive tumour-suppressor candidate for a p53-associated tumour-suppressor gene. In order to investigate the role of WAF1/CIP1 in the pathogenesis of primary human brain tumours we performed single-stranded conformation polymorphism (SSCP) analysis and direct sequencing of exon 2 of the gene in a representative series of 158 brain tumours and corresponding blood samples. In addition, all tumours were examined for mutations in exons 5-8 of the p53 gene. Analysis of WAF1/CIP1 revealed multiple polymorphisms, the most abundant being AGC-->AGA (Ser-->Arg) at codon 31 with an allele frequency of 8.5%. Less common polymorphisms included GTG-->GGG (Val-->Gly) at codon 25, GCC-->ACC (Ala-->Thr) at codon 64, CGC-->CTC (Arg-->Leu) at codon 32, GGC-->AGC (Gly-->Ser) at codon 14 and GCG-->GTG (Ala-->Val) at codon 39 each with an allele frequency of 0.3%. These polymorphisms were all located in a conserved region of exon 2. Two of the polymorphisms were also seen in a group of 157 healthy controls indicating that WAF1/CIP1 polymorphisms do not predispose to cancer. None of the tumours included in our series showed a somatic mutation in WAF1/CIP1. All samples were also analysed for loss of heterozygosity on the short arm of chromosome 6 in the region of the WAF1/CIP1 locus. Allelic loss was observed in only one patient with a glioblastoma. Mutations in the p53 gene were found in 22 of 158 tumours. No association was found between any polymorphism of the WAF1/CIP1 gene, p53 mutations and histopathological tumour type. Our data indicate that WAF1/CIP1 mutations are probably not involved in the formation of primary human brain tumours.     

Frequent occurrence of p53 mutations in rhabdomyosarcoma and leiomyosarcoma,but not in fibrosarcoma and malignant neural tumors

We have analyzed soft-tissue sarcomas (STS) molecularly for mutations in the tumor-suppressor gene p53 and immunohistochemically for expression of p53 and mdm2 proteins. In this study, tumor samples from 3 groups of soft-tissue sarcomas, i.e., fibrosarcomas, myogenic sarcomas and malignant neural tumors (MNT), were investigated. The methods applied encompass immunohistochemistry on 198 tumor samples using p53 antibodies (DO-1 and DO-7) and an mdm2 antibody (IF-2). Out of these, 100 samples were subjected to non-radioactive PCR-SSCP-sequencing analysis. Immunohistochemical detection rate for p53 (range of 57% to 67%) and for mdm2 proteins (range of 19 to 44%) was similar in all 3 groups. In higher tumor grades, an increased rate of immunopositivity was found for p53 but not for mdm2. Investigation of p53 mutational status revealed 6 mutations in myogenic sarcomas but none in malignant neural tumors or fibrosarcomas, suggesting different roles of p53 in the 3 STS groups. Interestingly, a G A transition in codon 245 (a CpG site) was found in 3 myogenic sarcomas. Our results and those of others suggest p53 codon 245 as a mutational hotspot in sarcomas, as recognized in carcinomas. © 1996 Wiley-Liss, Inc.     

Methylation of the hMLH1 promoter but no hMLH1 mutations in sporadic gastric carcinomas with high-level microsatellite instability

Microsatellite instability (MSI) in tumors from patients with hereditary non-polyposis colorectal cancer (HNPCC) is caused by germline mutations in mismatch repair (MMR) genes, principally hMSH2 and hMLH1. In contrast, somatic mutations in MMR genes are relatively rare in sporadic MSI(+) colon cancers. Rather, the majority of mutation-negative, MSI(+) cases involve hypermethylation of the hMLH1 promoter and subsequent lack of expression of hMLH1. The details of the mechanisms of this epigenetic gene silencing remain to be elucidated. In some colon cancer cell lines, hMLH1 promoter methylation is accompanied by mutation of 1 of the 2 alleles, whereas in other cell lines and tumors, such combinations have not been reported. To contribute to the characterization of MSI in gastric cancer and to directly investigate whether hMLH1 promoter methylation is accompanied by gene mutation in these cancers, we have analyzed 42 gastric tumors and corresponding normal tissue for MSI, hypermethylation of the hMLH1 promoter, and mutations in hMLH1 as well as hMSH2. We found that 10 (23.8%) of 42 cases of sporadic gastric cancer were MSI(+) and that 8 had at least 2 of 12 altered microsatellite loci. All samples with at least 2 altered loci exhibited methylation of the hMLH1 promoter region, but none had detectable mutations in hMLH1 or hMSH2. Our results confirm the importance of methylation of the hMLH1 promoter region in MSI(+) gastric tumors and suggest that methylation takes place in the absence of hMLH1 mutations in these tumors.     

p53 gene mutations occur in combination with 17p allelic deletions as late events in colorectal tumorigenesis

Coordinate loss of one copy of the p53 gene and mutation of the remaining copy occur in colorectal carcinomas and in many other human malignancies. However, the prevalence of p53 gene mutations in carcinomas which maintain both parental copies of p53 has not previously been evaluated. Moreover, it is not known whether p53 gene mutations are limited to malignant tumors or whether they can also occur in benign neoplasms. To answer these questions, a total of 58 colorectal tumors have been examined; in each tumor, allelic losses were assessed using restriction fragment length polymorphisms and p53 gene mutations were assessed by sequencing cloned polymerase chain reaction products. The following conclusions emerged: (a) p53 gene mutations occurred but were relatively rare in adenomas, regardless of size and whether the adenomas were derived from patients with familial adenomatous polyposis; (b) In carcinomas as well as in adenomas, p53 gene mutations were infrequently observed in tumors which contain both copies of chromosome 17p (17% of 30 tumors), while tumors which lost one copy of chromosome 17p usually had a mutation in the remaining p53 allele (86% of 28 tumors); (c) p53 gene mutations were found at similar frequencies in primary tumor samples and in cell lines derived from tumors. These and other data suggest that the rate limiting step in p53 inactivation is point mutation and that once a mutation occurs, loss of the remaining wild-type allele rapidly follows. Both mutations and allelic losses generally occur near the transition from benign to malignant growth, and the p53 gene may play a causal role in this progression.     

Mutational study of the 1p located genes p18ink4c, Patched-2, RIZ1 and KIF1B in oligodendrogliomas

Loss of 1p heterozygosity is one of the most characteristic events in oligodendrogliomas. Several genes located in this region have been previously studied to find the target gene implicated in the development of this tumor without success. Patched-2, RIZ1 and KIF1B are novel oncosuppressor genes located at 1p and involved in different kinds of tumors. We have studied these genes and p18(ink4c) using PCR/SSCP methods to detect sequence variations in a series of 40 oligodendrogliomas in which the allelic status at 1p was analyzed. Polymorphisms or no sequence changes were detected in all four genes analyzed. None of the genes analyzed seem to be the target-gene mapped at 1p involved by mutation in oligodendroglioma development.     

Loss of heterozygosity of 1p in uveal melanomas with monosomy 3

Gains and losses of chromosomes 1, 3, 6 and 8 are nonrandom chromosomal aberrations in uveal melanoma. Monosomy 3 is the most frequent abnormality and is associated with poor prognosis. To identify regions of allelic loss on the short arm of chromosome 1 and to investigate if these alterations contribute to uveal melanoma progression, we performed microsatellite analysis of 10 loci in 70 uveal melanomas. A total of 51 tumors were obtained from patients with clinical follow-up data, 19 tumors were from recent patients without follow-up. Loss of heterozygosity (LOH) of at least 1 marker was more frequent in tumors with monosomy 3 (40%) than in tumors with disomy 3 (10%). In particular, loss of the entire short arm of chromosome 1 was only observed in tumors with monosomy 3 (p = 0.0001). By comparing the extent of 1p LOH in all tumors with monosomy 3, we were able to define a smallest region of overlap (SRO) of approximately 55 Mb, which is flanked by markers D1S507 and D1S198. On the basis of our data and published cytogenetic data, we propose that 1p31 harbors genes involved in the progression of uveal melanoma with monosomy 3.     

P16 gene deletions and point mutations in patients with agnogenic myeloid metaplasia (AMM).

Studies of p16 alterations with homozygous deletions and mutation analysis were done in 32 patients with agnogenic myeloid metaplasia (AMM) including six patients in leukemic phase. No homozygous deletions were found and, one patient was found to have a shift band in exon 2C fragment by PCR-SSCP analysis. Further sequence analysis demonstrated that the mutated band was a point mutation of G to A in exon 2 codon 140 (GCG-->ACG) causing an amino acid substitution of alanine to threonine demonstrating this patient either carried an mutated gene in one allele as a polymorphism (heterozygous carrier of a mutant p16 gene) or carried a mutant p16 gene clone. This study demonstrates that p16 alterations with homozygous deletions and mutations were very rare in patients with AMM. A single patient found to have a shifted band by PCR-SSCP may be represented as a coincidence or as a polymorphism with a heterozygous carrier of mutated p16 gene, predisposable to AMM or as a mutant p16 gene which can be infrequently observed in this disease.     

Chromosome 10 and 17 deletions and p53 gene mutations in Thai patients with astrocytomas

The tumor suppressor gene locus is known to be partly responsible for the tumorigenesis of sporadic gliomas, but the genetic events that drive the neoplastic process of this tumor remain largely unknown. We correlated the results of loss of heterozygosity (LOH) analysis on chromosomes 10 and 17 and a point mutation analysis of a tumor suppressor gene, p53, in 21 patients with astrocytomas at different stages. LOH was determined in tumor and leukocyte DNAs of primary human central nervous system tumors. The incidence rate of brain tumors corresponded to every p53-coding exon for single-strand conformation polymorphisms (SSCP) and the mutations were confirmed by sequencing. p53 mutations were found in 2 of 10 glioblastomas (20%) and in 1 of 8 low-grade astrocytomas (12.5%). Similarly, LOH on chromosome 10 was also found in 2 of 10 glioblastomas (20%) and 1 of 8 low-grade astocytomas (12.5%). Neither of the p53 mutations nor LOH on chromosome 10 was observed together in the tumor types analyzed. Interestingly, the p53 mutations were found in 29% of patients with LOH on chromosome 17. The fact that p53 mutation and LOH on chromosome 17 were found together only in glioblastomas, suggested that these genetic changes may accumulate during astrocytoma progression.     

1p36 is a preferential target of chromosome 1 deletions in astrocytic tumours and homozygously deleted in a subset of glioblastomas

Astrocytic, oligodendroglial and mixed gliomas are the commonest gliomas in adults. They have distinct phenotypes and clinical courses, but as they exist as a continuous histological spectrum, differentiating them can be difficult. Co-deletions of total 1p and 19q are found in the majority of oligodendrogliomas and considered as a diagnostic marker and a prognostic indicator. The 1p status of astrocytomas has not yet been thoroughly examined. Using a chromosome 1 tile path array, we investigated 108 adult astrocytic tumours for copy number alterations. Total 1p deletions were rare (2%), however partial deletions involving 1p36 were frequently identified in anaplastic astrocytomas (22%) and glioblastomas (34%). Multivariate analysis showed that patients with total 1p deletions had significantly longer survival (P=0.005). In nine glioblastomas homozygous deletions at 1p36 were identified. No somatic mutations were found among the five genes located in the homozygously deleted region. However, the CpG island of TNFRSF9 was hypermethylated in 19% of astrocytic tumours and 87% of glioma cell lines. TNFRSF9 expression was upregulated after demethylation of glioma cell lines. Akt3 amplifications were found in four glioblastomas. Our results indicate that 1p deletions are common anaplastic astrocytomas and glioblastomas but are distinct from the 1p abnormalities in oligodendrogliomas.