MDM2 gene amplification and transcript levels in human sarcomas: relationship to TP53 gene status.

BACKGROUND: Alterations of the TP53 tumor suppressor gene appear to be implicated in the tumorigenesis and progression of several types of human cancer, including different histologic subtypes of sarcomas. The MDM2 (murine double minute-2) gene encodes a nuclear phosphoprotein that may interact with both mutant and wild-type p53 proteins, thereby inhibiting p53-mediated transactivation in a dose-dependent manner. Recently it has been suggested that mdm2 and p53 proteins are components of an autoregulatory loop in which the MDM2 gene is transactivated by p53. PURPOSE: Our purpose was to examine the frequency of MDM2 amplifications in larger panels of sarcomas, determine if the mRNA level could be elevated in tumors without concomitant gene amplification, and relate MDM2 findings to the TP53 status of the tumors. METHODS: Sarcoma tissue of different histologic subtypes was obtained from 68 patients at the time of surgery and from 26 human xenografts in nude mice. In addition, two human sarcoma cell lines (OSA and U2OS) were studied. Genomic DNA from tumor tissue, in vitro cell lines, and peripheral blood cells were isolated by Southern-blot analysis methods to determine MDM2 gene amplification. Tumor DNA was analyzed for possible TP53 gene mutations in exons 5, 7, and 8 by constant denaturing gel electrophoresis. To determine the MDM2 and TP53 mRNA levels, Northern-blot analysis was performed. RESULTS: Amplification of the MDM2 gene was detected in 10 tumors (10.3%). Whereas MDM2 amplification and/or over-expression were found only in two (U2OS and OSA cell lines) of 18 osteosarcomas, one of 20 malignant fibrous histiocytomas (MFHs), and in none of 14 leiomyosarcomas, such alterations were observed in two of two fibrosarcomas, three of six malignant schwannomas, three of 19 liposarcomas, and in the one hemangiopericytoma examined. MDM2 overexpression was found in all nine examined cases with and in three tumors without amplification. TP53 mutations were detected in 12 cases (five osteosarcomas, four MFHs, and three leiomyosarcomas), of which none showed amplification, but one had increased levels of MDM2 mRNA. None of the fibrosarcomas, malignant schwannomas, and liposarcomas examined had mutated TP53. The six sarcomas that showed high TP53 mRNA expression in the absence of gene mutation also had elevated levels of MDM2 mRNA. CONCLUSIONS: The present data provide further indications that increased MDM2 expression level, caused by gene amplification or altered regulation of transcription, is involved in tumor progression of some, but not all, sarcoma subtypes.

HMGIC, the gene for an architectural transcription factor, is amplified and rearranged in a subset of human sarcomas.

Amplified segments of the long arm of chromosome 12 are frequently observed in human sarcomas. In most cases there are separate amplified regions around the MDM2 and CDK4 genes. Here we show recurrent amplification of a third region encompassing HMGIC, a human architectural transcription factor gene. Reduced amplification frequency of sequences flanking the gene was observed, indicating that inclusion of this third region in the amplicons is also selected for. In three samples only the 5' part of HMGIC was amplified, suggesting preferential loss of the 3' part of the gene preceding or during amplification. In several other samples rearrangement of the gene was observed. Expression analysis showed transcripts of aberrant sizes, lacking 3' sequences, and 3' RACE of one sample revealed replacement of exons 4 and 5 with ectopic sequences. This truncation of HMGIC resembles that reported for translocations of HMGIC in benign tumors, including lipomas, and it is striking that the gene was frequently amplified or rearranged in well differentiated liposarcomas, the malignant counterpart of lipomas. It seems conceivable that high levels of either full length or truncated hmgic could be relevant for the etiology of these tumors.

Amplification and overexpression of PRUNE in human sarcomas and breast carcinomas-a possible mechanism for altering the nm23-H1 activity.

PRUNE, the human homologue of the Drosophila gene, is located in 1q21.3, a region highly amplified in human sarcomas, malignant tumours of mesenchymal origin. Prune protein interacts with the metastasis suppressor nm23-H1, but shows impaired affinity towards the nm23-H1 S120G mutant associated with advanced neuroblastoma. Based on these observations, we previously suggested that prune may act as a negative regulator of nm23-H1 activity. We found amplification of PRUNE in aggressive sarcoma subtypes, such as leiomyosarcomas and malignant fibrous histiocytomas (MFH) as well as in the less malignant liposarcomas. PRUNE amplification was generally accompanied by high mRNA and moderate to high protein levels. The sarcoma samples expressed nm23-H1 mostly at low or moderate levels, whereas mRNA and protein levels were moderate to high in breast carcinomas. For the more aggressive sarcoma subtypes, 9/13 patients with PRUNE amplification developed metastases. A similar situation was observed in all breast carcinomas with amplification of PRUNE. Infection of NIH3T3 cells with a PRUNE recombinant retrovirus increased cell proliferation. Possibly, amplification and overexpression of PRUNE has the same effect in the tumours. We suggest that amplification and overexpression of PRUNE could be a mechanism for inhibition of nm23-H1 activity that affect the development or progression of these tumours.

Identification of two distinct chromosome 12-derived amplification units in neuroblastoma cell line NGP.

The neuroblastoma cell line NGP contains two homogeneously staining regions (hsr). One of these hsrs contains MYCN sequences. Reverse painting experiments demonstrated that the second HSR consisted of two chromosome 12-derived amplification units, located at 12q14-15 and 12q24. Southern blot and fluorescence in situ hybridization (FISH) analysis showed amplification of genes located at 12q14-15: SAS, MDM2, and CDK4, GLI, CHOP, CDK2, and A2MR were found not to be amplified. FISH further demonstrated amplification of RSN, a gene located at 12q24. The finding of two distinct chromosome 12 amplification units in a neuroblastoma cell line NGP is reminiscent of recent findings in well-differentiated liposarcoma (WDLPS) and other sarcomas. The second amplification unit on chromosome 12 in NGP is located more distal (12q24) than the one observed in WDLPS (12q21). The mechanism and biologic significance of this amplification process in neuroblastoma and WDLPS remain to be elucidated.

The protooncogene CHOP/GADD153, involved in growth arrest and DNA damage response, is amplified in a subset of human sarcomas.

The C/EBP-homologous transcription factor CHOP (GADD153) is inducible by growth inhibition or DNA damage, and has been shown to be oncogenically activated by the specific (12;16) translocation in human myxoid liposarcoma. We have now found CHOP amplification in two sarcoma cell lines with previously reported amplification of the nearby GLI gene. Among 98 other human sarcomas of various types, CHOP was amplified in a hemangiopericytoma, a liposarcoma, and two osteosarcoma. High constitutive expression levels of CHOP were observed in tumors with gene amplification, but also in some other samples. The nearby MDM2 gene, which codes for a protein that may inactivate wild-type p53, has previously been reported to be frequently amplified in sarcoma. In our sarcoma panel, MDM2 was amplified in 9 cases. MDM2 and CHOP were co-amplified in two of these, whereas the two osteosarcomas had amplified CHOP but not MDM2. CHOP was amplified in both cell lines with GLI amplification, and MDM2 only in one. No mutations in the TP53 gene have been found in samples with amplification of MDM2. In contrast, the cell line in which CHOP but not MDM2 was amplified had mutated TP53, suggesting that selection of this amplicon was not mediated through p53 inactivation.

Ring chromosomes in a malignant mesenchymoma.

We report, for the first time, the cytogenetic and molecular genetic constitution of a human mesenchymoma. As in several other soft tissue sarcomas, supernumerary ring and rod-shaped marker chromosomes were observed next to an otherwise normal diploid karyotype. Comparative genomic in situ hybridization and whole chromosome painting experiments revealed that chromosome 1q21-q25 and 12q14-q15 sequences were amplified, and that these sequences resided on the supernumerary marker chromosomes. We assume that, in this malignant mesenchymoma, the observed chromosomal anomalies may be associated with its well differentiated liposarcomatous component.

Dedifferentiation of a well-differentiated liposarcoma to a highly malignant metastatic osteosarcoma: amplification of 12q14 at all stages and gain of 1q22-q24 associated with metastases.

Well-differentiated liposarcomas (WDLPS), especially those located in the retroperitoneum, may occasionally undergo dedifferentiation. Although this process is associated with a more aggressive clinical course, dedifferentiated liposarcomas rarely produces metastases. The case reported here is rather uncommon: A retroperitoneal WDLPS gave lung metastases that were diagnosed as highly malignant osteosarcomas. We used comparative genomic hybridization (CGH), fluorescence in situ hybridization (FISH), and Southern blot analyses to characterize the copy number changes and genetic aberrations occurring at different stages of the disease. In the primary tumor, the only detectable aberration was amplification of 12q13-q14, which was present only in a fraction of the cells and revealed by FISH analysis. High-level amplification of 12q13-q14, involving CDK4, MDM2, and HMGIC, was seen both in the relapse and the metastases. The second most common change, gain or high-level amplification of 1q22-q24, was detectable by CGH only in the osteogenic metastases, as was loss of the distal 2q. FISH analyses revealed considerable heterogeneity in the samples, and the percentage of cells showing aberrations was significantly higher in the metastatic samples. In particular, increased copy numbers of 789f2, a marker for 1q21 amplification in sarcomas, was observed in more than 65% of the cells in the metastatic samples, but in less than 10% of the cells from the recurrent samples. These observations could indicate that 1q amplification, in particular, may be indicative of a more malignant phenotype and ability of metastasis in WDLPS, as has also been suggested by others.

A well-differentiated liposarcoma with a new type of chromosome 12-derived markers.

Well-differentiated liposarcomas (WDLPS) are cytogenetically characterized by the presence of supernumerary ring or giant rod marker chromosomes. These supernumerary chromosomes are composed of amplified sequences from chromosome 12 (12q14 approximately 15) in association with amplified segments from various other chromosomes, and contain alterations of the alpha satellite sequences. We report a case of WDLPS of the lipoma-like and sclerosing subtype that contains a novel type of supernumerary marker chromosome. Instead of rings or giant rods, these cells had three apparently identical copies of a subtelocentric supernumerary marker with a size and shape similar to C-group chromosomes. Fluorescence in situ hybridization analysis revealed that the markers were composed of amplified material from 12q14 approximately 15, including the genes MDM2 and CDK4. Similar to the rings and giant rods observed in other WDLPS cases, these unusual markers had no alpha satellite repeats at the primary constriction site, but centromeric activity could be demonstrated by using anti-centromere protein C antibodies. These findings show that the supernumerary markers of WDLPS may be variable in size and shape, but consistently share the same genomic structure, specifically 12q amplified sequences together with centromere alterations, and underline the importance of molecular methods in the diagnosis of adipose tissue tumors.

Amplification of chromosome subregion 12p11.2-p12.1 in a metastasis of an i(12p)-negative seminoma: relationship to tumor progression?

Cytogenetic analysis of a metastasis of a human testicular germ cell tumor (seminoma) revealed multiple numerical and structural anomalies, including an abnormally banding region (ABR) present on the short arm of one of the chromosome 12 homologs. Fluorescence in situ- and comparative genomic hybridization experiments revealed that the ABR results from the amplification of 12p11.2-p12.1 derived sequences. We speculate that this particular region may harbor gene(s) relevant for testicular germ cell tumor progression.

Frequent loss of 9p21 (p16(INK4A)) and other genomic imbalances in human malignant fibrous histiocytoma.

To search for new recurrent genetic aberrations in malignant fibrous histiocytoma (MFH), a combination of conventional cytogenetic, comparative genomic hybridization (CGH), and Southern blot analyses was applied to a series of 34 tumors. Cytogenetic analysis revealed the presence of multiple structural and numerical aberrations, including marker chromosomes, telomeric associations, double minutes, and ring chromosomes. The most frequent genomic imbalances in this series of neoplasms as detected by CGH were gains of 1q21-q22 (69%), 17q23-qter (41%), and 20q (66%), and losses of 9p21-pter (55%), 10q (48%), 11q23-qter (55%), and 13q10-q31 (55%). Southern blot analyses with p16(INK4A) (CDKN2A; 9p21) and RB1 (13q14) probes provided clear indications for frequent deletions of these tumor suppressor genes, and as such, substantiated the CGH results. Additionally, examination of the TP53 and MDM2 genes showed frequent loss and amplification, respectively. These data indicate that genes involved in the RB1- and TP53-associated cell cycle regulatory pathways may play prominent roles in the development of human MFH.

Deregulation of D-type cyclins in uterine cancers. Cyclin D1/D3 is differentially expressed in cervical cancer.

BACKGROUND: We have previously found cyclin D1 to be overexpressed in 45% of corpus cancers, but in only 3% of cervical cancers. To see whether and how D-type cyclins contribute to the neoplastic phenotype in uterine cancers, aberrant expression and association between cyclins D1 and D3 proteins were studied. MATERIALS AND METHODS: Expression of cyclin D3 was investigated by immunohistochemistry in 51 patients with primary cancer of the corpus and 73 cases of primary cervical carcinomas. Amplification of the cyclin D1 and D3 genes was investigated by fluorescence in situ hybridisation (FISH). RESULTS: We found high/moderate levels of cyclin D3 in 53.5% of cervical cancers and 55% of corpus cancers. High/moderate expression of cyclin D1 and D3 was associated in the corpus cancers, but not in cervical cancers. The accumulation of cyclin D3 but not D1 protein could, in some cases, be explained by increased gene dosage since extra copies of chromosome 6 were found. CONCLUSION: This study demonstrates that in cervical cancers cyclin D3 may compensate for low levels of cyclin D1, whereas in corpus cancers both isoforms may contribute to the neoplastic phenotype.

A physical map of a 1.3-Mb region on the long arm of chromosome 12, spanning the GLI and LRP loci.

We have used pulsed-field gel electrophoresis to construct a long-range restriction map spanning more than 1.3 million bp of the q13-q14 segment of chromosome 12. Within this region lie the genes coding for the gli oncogene and the low-density lipoprotein receptor-related protein (LRP). The distance between the genes is about 200-300 kb. We also observe a methylation-free island 3' to the LRP gene.

Yeast TFIIIA + TFIIIC/tau-factor, but not yeast TFIIIA alone, interacts with the Xenopus 5S rRNA gene.

The successful use of mixed heterologous in vitro transcription systems has suggested that the species specificity of RNA polymerase III transcription is low. To see if this extends to lower eukaryotic class III transcription factors, we compared the interactions of the two yeast assembly factors, TFIIIA and TFIIIC/tau factor, with a homologous yeast 5S rRNA gene and a heterologous Xenopus laevis somatic 5S rRNA gene. Transcription assays showed that the Xenopus gene was transcriptionally inactive in a crude cell-free yeast extract that actively transcribes the homologous gene. However, the Xenopus gene was still able to compete for limiting transcription factors. Electrophoretic DNA binding assays revealed that while TFIIIA bound avidly to the yeast gene (generating the 'A-complex'), it had no affinity for the Xenopus 5S rRNA gene. Nevertheless, a complex of both TFIIIA and TFIIIC/tau factor (the 'AC-complex') was formed on the two genes with similar affinity, although only the complex assembled on the homologous gene was able to activate transcription. Thus enough sequence information is present on the heterologous gene to direct transcription factor assembly, but not to activate transcription. Like its counterpart in Xenopus, the yeast TFIIIA appears to be a zinc binding protein that is inactivated by EDTA and 1,10-phenanthroline, and reactivated in the presence of zinc ions. Bound to the 5S rRNA gene, TFIIIA is however significantly more resistant to inactivation by chelators than in its free state. The AC-complex differs from the A-complex by being less affected by chelators, and by being more sensitive to the dissociating effect of single-stranded DNA.

Sensitive fluorescent in situ hybridisation method for the characterisation of breast cancer cells in bone marrow aspirates.

AIM: The presence of malignant cells in the blood and bone marrow of patients with cancer at the time of surgery may be indicative of early relapse. In addition to their numbers, the phenotypes of the micrometastatic cells might be essential in determining whether overt metastases will develop. This study aimed to establish a sensitive method for the detection and characterisation of malignant cells present in bone marrow. METHODS: In spiking experiments, SKBR3 cells were mixed with mononuclear cells in known proportions to mimic bone marrow samples with micrometastatic cells. Tumour cells were extracted using SAM-M450 Dynabeads coupled to the MOC-31 anti-epithelial antibody, and were further analysed for amplification of erbB2 and int2 by fluorescent in situ hybridisation (FISH). erbB2 and int2 copy numbers were also determined in 15 primary breast cancers, and bone marrow samples from patients with amplification were analysed for micrometastatic cells by immunomagnetic enrichment and FISH. RESULTS: In model experiments, cells with amplification could be detected in bead selected fractions when ratios of tumour cells (SKBR3) to mononuclear cells were as low as 10:10(7). Among the tumour samples, eight showed increased copy numbers of erbB2 and/or int2, and three of these patients had detectable numbers of tumour cells in their bone marrow: 4000, 540, and 26 tumour cells/10(7) mononuclear cells, respectively. The patient with 540 tumour cells/10(7) mononuclear cells showed high level amplification of erbB2 and suffered from a particularly aggressive disease, whereas the patient with 4000 tumour cells/10(7) mononuclear cells had favourable disease progression. CONCLUSION: These results demonstrate the feasibility and advantage of combining immunomagnetic selection and FISH characterisation of cancer cells in bone marrow samples. It is possible that molecular characterisation of such cells could provide prognostically valuable information.

[Microarray technology--potential in cancer research]. / Mikromatriser i kreftforskning--nå trenger vi ikke lete bare under gatelyktene!

BACKGROUND: Researchers have worked for decades to solve the enigma of cancer. We know that essential checkpoints in the life cycle of cells have to be disrupted in order to create a tumour cell, and some of the genes and proteins involved have been identified. Most of the previous work on identifying these genes have been based on "educated guesswork", as the methods and technologies used have been limited to the examination of genes one by one, or a few at a time. MATERIAL AND METHODS: Microarray technology allows tens of thousands of genes to be examined at the same time, without any previous information on the genes. Both expression levels and copy numbers of the genes can be evaluated. Our studies of breast cancer and bone tumours are presented, as well as examples from the literature. RESULTS AND DISCUSSION: Microarray analyses have been used to produce molecular portraits of breast cancer, malignant melanomas and other cancers. These portraits may help in sub-classification of tumours, in prognosis, and in the general understanding of cancer. For example, studies of gene expression patterns of breast carcinomas, similar with respect to classic prognostic markers (such as ER status, grading and morphology), have identified subgroups of patients that show differences in survival.

Separate amplified regions encompassing CDK4 and MDM2 in human sarcomas.

Amplification of MDM2 and CDK4 is observed frequently in human sarcomas. Although overexpression of these protooncogenes might inhibit growth regulation through the TP53- and retinoblastoma tumor suppressor protein (RB)-mediated pathways, neither gene was included consistently in all of the amplicons observed in our sarcoma panel. It was unclear whether both of these genes were selected for during amplification. Furthermore, in some samples without amplification of MDM2 or CDK4, comparative genomic hybridization showed amplification in the 12q13-15 region, suggesting that another selection mechanism might also be involved. To investigate the possibility that another target gene, which may be located between CDK4 and MDM2, could be the driving force, we characterized the involvement of 17 loci from this region in 12q13-15 amplicons that were detected previously in 21 sarcoma samples. The results showed discrete amplicons around MDM2 and CDK4 with reduced amplification of the intervening sequences. This suggests that there is separate selection for amplification of the two genes, and it makes the possibility of a common selective gene unlikely. Furthermore, D12S8, localized distal to MDM2, was amplified almost as frequently as MDM2 and was also amplified in one of the samples without MDM2 or CDK4 amplification. The data suggest that amplification of at least three different regions within the 12q13-15 segment may be selected for in tumor cells involving MDM2, CDK4, or a more distally located gene, possibly near D12S8.

Mapping of amplification units in the q13-14 region of chromosome 12 in human sarcomas: some amplica do not include MDM2.

Amplification of cellular oncogenes may be important for the development and progression of malignant tumors. In human sarcomas, amplification of several genes located to the q13-14 region of chromosome 12 has been reported. Because the mdm2 protein seems to inactivate the tumor suppressor protein p53, a selective growth advantage of 12q13-14 amplification has previously been assigned to increased copy number and expression of the MDM2 gene. We have analyzed a panel of 98 human sarcomas of different subtypes to characterize the 12q13-14 amplica and determine which of the genes GLI, A2MR, SAS, MDM2, and GADD153 (CHOP) in this region was most consistently amplified. MDM2 was amplified in 9 of the tumors, SAS in 10, GADD153 in 4, GLI in 2, and A2MR in 2. Amplification was, in most cases, associated with increased expression of the corresponding gene. SAS and MDM2 were coamplified in 8 of the tumors, whereas GADD153, GLI, and A2MR were only amplified together with SAS. One liposarcoma showed amplification of MDM2 alone, whereas two osteosarcomas and a rhabdomyosarcoma cell line showed amplification of SAS and GADD153 (CHOP) but not MDM2. It is suggested that the selective target for these amplica may be an as yet unidentified gene localized between SAS and MDM2.

Comparative genomic hybridization analysis of human sarcomas: II. Identification of novel amplicons at 6p and 17p in osteosarcomas.

Using comparative genomic hybridization (CGH), we have identified and mapped regions of DNA amplification in primary and metastatic osteosarcomas. Samples were obtained from four patients and ten independent xenografts. Sixty-four percent of the tumors showed increased DNA-sequence copy numbers, affecting 23 different chromosomal sites. Most of these regions were not previously associated with the development and/or progression of these tumors. Amplicons originating from 1q21-q23, 6p, 8q23-qter, and 17p11-p12 were observed most frequently. The 6p and 17p11-p12 amplicons seem to be specific for osteosarcomas, indicating that these regions may harbor genes relevant for the development of these tumors.

Comparative genomic hybridization analysis of human sarcomas: I. Occurrence of genomic imbalances and identification of a novel major amplicon at 1q21-q22 in soft tissue sarcomas.

Comparative genomic hybridization (CGH) was recently developed as a tool to survey entire genomes for variations in DNA sequence copy numbers. We have applied this technique to detect and map amplified regions in 54 soft tissue sarcomas. Aberrations were detected by visual analysis of hybridizations or contrast-enhanced digital images, followed by quantitative digital ratio imaging of the aberrant chromosomes. Several tumors showed increased DNA sequence copy number at 12q14, as expected. However, CGH analysis detected amplification of 12q14 also in some tumors where neither MDM2 nor CDK4 was amplified, suggesting that another as yet unknown gene(s) may drive amplification of this region in sarcomas. Furthermore, a novel recurring amplicon was detected at 1q21-q22. DNA amplifications coinciding with this segment were as frequent as those observed for 12q14, indicating that 1q21-q22-linked gene(s) may also play an important role in the development and/or progression of human soft tissue sarcomas.