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 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 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.

Ectopic sequences from truncated HMGIC in liposarcomas are derived from various amplified chromosomal regions.

The HMGIC gene codes for an architectural transcription factor frequently rearranged by translocation in lipomas and other benign mesenchymal tumors. In sarcomas, malignant tumors of mesenchymal origin, the gene is also found to be rearranged, but in addition amplified and overexpressed. Here we report the sequence, chromosomal localization, and expression patterns of 11 novel ectopic sequences fused to exons 2 and 3 of HMGIC in seven different sarcoma samples. In addition, we identified a number of variant transcripts observed previously in benign tumors. Consistent with the suggested role of HMGIC in adipocytic differentiation, most of the novel ectopic sequences were observed in well-differentiated liposarcomas. These tumors are known to have complex marker chromosomes containing amplified segments from several chromosomes. Five novel sequences were derived from 12q14-q15, where HMGIC resides, two from 1q24, a region frequently amplified in these types of tumors, two from 11q14, and one from chromosome 2. All except one of the aberrant transcripts encoded truncated proteins with intact DNA-binding domains (AT hooks) but lacking the C-terminal acidic region, a target for constitutive phosphorylation by protein kinase CK2. Some of the ectopic sequences were transcribed in other tissues, and most of the ectopic sequences also showed recurrent amplification in liposarcomas.

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.

[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.

Characterization of centromere alterations in liposarcomas.

Supernumerary ring and large marker chromosomes are a characteristic of atypical lipomas and well-differentiated liposarcomas (ALP-WDLPS) and are composed of amplified 12q14-15 sequences in association with variable segments from other chromosomes. Although stably transmitted, these chromosomes contain centromeric alterations, showing no detectable alpha-satellite sequences. We performed C-banding, fluorescence in situ hybridization, and immunostaining with anti-centromere antibodies in 8 cases of liposarcomas with supernumerary rings and large markers, including 5 ALP-WDLPS and 3 dedifferentiated-LPS and high-grade LPS. Our results with alpha-satellite probes and anti-CENPB antibodies confirm the lack of detectable alpha-satellite sequences in the five ALP-WDLPS supernumerary chromosomes, whereas centromeric activity was proved by the detection of kinetochores by using anti-CENPC antibodies. In contrast, the high grade and dedifferentiated liposarcomas showed a different pattern. In 2 cases, amplified chromosome 12 sequences, including amplification of alpha-satellite 12 sequences in 1 case, were present on chromosomes with typical centromeres. In another case, the rings were similar to WDLPS-ALP rings, but a large marker contained a chromosome 5 centromere and amplified alpha-satellite sequences from chromosome 8. ALP-WDLPS is the first example of a tumor class for which the presence of stable analphoid chromosomes is a constant and specific abnormality. Formation of newly derived centromeres, so-called neocentromeres, could be an original and effective way to maintain a selective advantage in neoplastic cells by conferring stability to the supernumerary chromosomes of ALP-WDLPS. The activation of normally non-centromeric sequences might be obtained by an epigenetic mechanism due to the peculiar chromatin conformation of these highly complex chromosomes.

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.

A novel chromosomal region of allelic loss, 4q32-q34, in human osteosarcomas revealed by representational difference analysis.

Representational difference analysis (RDA) of a human osteosarcoma xenograft resulted in the isolation of four tumor-associated homozygously deleted DNA fragments, all originating from chromosome 4, region q32-q34. Southern blot analysis using the RDA fragments and interphase FISH analysis using PACs corresponding to these RDA fragments revealed allelic loss of the 4q32-q34 region in 17 of 27 (63%) osteosarcomas tested. These results suggest the involvement of tumor suppressor gene(s) within this chromosomal region in osteosarcoma development. The RDA fragments and corresponding PAC clones will be instrumental in the isolation of such gene(s). Genes Chromosomes Cancer 26:115-124, 1999.

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.

Characterization of the 17p amplicon in human sarcomas: microsatellite marker analysis.

The structure of the 17p amplicon from 9 human sarcoma specimens evaluated by comparative genomic hybridization (CGH) has been studied by analyzing 28 microsatellite markers by PCR. Eleven sarcoma specimens showing no DNA copy number increases at 17p by CGH were analyzed as control samples. Five specimens were analyzed by Southern blotting using probes that have previously shown amplification at the 17p12 region in astrocytoma and high-grade osteosarcoma samples. Microsatellite marker analyses revealed that all samples but 1 showing copy number increases at 17p by CGH displayed allelic imbalance that confirmed the CGH findings. Seven of these 9 cases displayed gain in copy number by microsatellite marker analysis. Four cases displaying gain in copy number were associated with loss of heterozygosity at other loci. Southern blot analysis showed amplification in 3 cases, all of them had shown copy number increases by CGH and microsatellite marker analysis, except one case, which was not included in the microsatellite marker analysis. Our results reveal the complexity of the 17p amplicon in sarcomas, suggesting that multiple target genes are involved in tumorigenesis.

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.

Structure of the supernumerary ring and giant rod chromosomes in adipose tissue tumors.

Supernumerary ring or giant rod marker chromosomes are a characteristic of well-differentiated liposarcomas (WDLPS) and atypical lipomas (ALP) and are often observed as the sole cytogenetic abnormality, but are rare in lipomas. Using a combination of different methods, we extensively investigated the structure and composition of rings and giant rods in a series of 17 WDLPS-ALP samples and three intra- or intermuscular lipomas (IMLP), revealing a unique combination of particular features strikingly related to these tumors. Although the rings and rods displayed in vitro and in vivo stability, the presence of alpha-satellites could not be detected on these supernumerary structures. Comparative genomic hybridization analysis, in combination with fluorescence in situ hybridization, identified the chromosomal regions contributing to the formation of these chromosomes: in WDLPS-ALP, all carried amplifications of 12q 14-15 and the MDM2 gene, with variable other noncontiguous regions. In the three IMLP, the rings consistently carried amplifications of 12q15-21 and 1q21, but increased copies of MDM2 were found in only one case. Other genes located more proximal in 12q14-15 were amplified in several WDLPS-ALP, but showed a normal copy number in IMLP. Furthermore, the immunohistochemical expression of the MDM2 protein was detected in most (12/14) WDLPS-ALP, in 1-30% of the cells, but never in IMLP. These supernumerary chromosomes represent a peculiar kind of amplification structure, midway between double minute chromosomes and homogeneously staining regions, but the mechanisms underlying the formation of these structures remain obscure.

Molecular characterization of a novel amplicon at 1q21-q22 frequently observed in human sarcomas.

In a recent comparative genomic hybridization (CGH) study of a panel of sarcomas, we detected recurrent amplification of 1q21-q22 in soft tissue and bone tumours. Amplification of this region had not previously been associated with sarcoma development, but occasional amplification of CACY/S100A6 and MUC1 in 1q21 had been reported for melanoma and breast carcinoma respectively. Initial screening by Southern blot analysis showed amplification of S100A6, FLG and SPRR3 in several sarcomas and, in a first attempt to characterize the 1q21-q22 amplicon in more detail, we have now investigated the amplification status of these and 11 other markers in the region in 35 sarcoma samples. FLG was the most frequently amplified gene, and the markers located in the same 4.5-Mb region as FLG showed a higher incidence of amplification than the more distal ones. However, for most of the 14 markers, amplification levels were low, and only APOA2 and the anonymous marker D1S3620 showed high-level amplifications (> tenfold increases) in one sample each. We used fluorescence in situ hybridization (FISH) to determine the amplification patterns of two overlapping yeast artificial chromosomes (YACs) covering the region between D1S3620 and FLG (789f2 and 764a1), as well as two more distally located YACs in nine selected samples. Six samples had amplification of the YAC containing D1S3620 and, in three, 764a1 was also included. Five of these tumours showed normal copies of the more distal YACs; thus, it seems likely that an important gene may be located within 789f2, or very close. Two samples had high copy numbers of the most distal YACs. Taken together, FISH and molecular analyses indicate complex amplification patterns in 1q21-q22 with at least two amplicons: one located near D1S3620/789f2 and one more distal.

Ectopic expression of target genes may represent an inherent limitation of RT-PCR assays used for micrometastasis detection: studies on the epithelial glycoprotein gene EGP-2.

Our objective was to develop and study the feasibility of a quantitative, nested reverse-transcription polymerase chain reaction (RT-PCR) assay for detection of micrometastatic, epithelial tumor cells using the epithelial glycoprotein EGP-2 gene as a target. Several carcinoma cell lines and peripheral blood samples of 10 healthy volunteers were screened for levels of EGP-2 mRNA. The assay included EGP-2 competitor molecules, carrying an internal deletion, that had been titrated by limiting dilution. Seven carcinoma cell lines showed a wide spectrum of EGP-2 mRNA expression levels, with the highest values (20-100 molecules/cell) seen in 3 breast-cancer cell lines. Unexpectedly, a consistent low level of EGP-2 mRNA expression (0.0004 molecules/cell) was observed in peripheral blood mononuclear cells, probably representing ectopic non-functional expression. Because of this background level, spiking experiments with T47D breast-carcinoma cells added to blood mononuclear cells exhibited a detection limit that was not better than approximately one tumor cell in 2 x 10(4) normal cells. Together with the considerable variation of EGP-2 transcript levels that is observed in different carcinoma cell lines, the extent of expression in normal blood cells would prevent a reliable estimation of low numbers of carcinoma cells in clinical samples. A similar situation might well apply for other target genes. This emphasizes the need for a critical evaluation of the different steps involved in the methods used for RT-PCR detection of micrometastatic tumor cells.

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.

Recurrent gains of 1q, 8 and 12 in the Ewing family of tumours by comparative genomic hybridization.

Comparative genomic hybridization (CGH) was used to detect copy number changes of DNA sequences in the Ewing family of tumours (ET). We analysed 20 samples from 17 patients. Fifteen tumours (75%) showed copy number changes. Gains of DNA sequences were much more frequent than losses, the majority of the gains affecting whole chromosomes or whole chromosome arms. Recurrent findings included copy number increases for chromosomes 8 (seven out of 20 samples; 35%), 1q (five samples; 25%) and 12 (five samples; 25%). The minimal common regions of these gains were the whole chromosomes 8 and 12, and 1q21-22. High-level amplifications affected 8q13-24, 1q and 1q21-22, each once. Southern blot analysis of the specimen with high-level amplification at 1q21-22 showed an amplification of FLG and SPRR3, both mapped to this region. All cases with a gain of chromosome 12 simultaneously showed a gain of chromosome 8. Comparison of CGH findings with cytogenetic analysis of the same tumours and previous cytogenetic reports of ET showed, in general, concordant results. In conclusion, our findings confirm that secondary changes, which may have prognostic significance in ET, are trisomy 8, trisomy 12 and a gain of DNA sequences in 1q.