Ring chromosomes and/or giant marker chromosomes have been observed in a variety of human tumor types, but they are particularly common in a subgroup of mesenchymal tumors of low-grade or borderline malignancy. These rings and markers have been shown to contain amplified material predominantly from 12q13–15, but also sequences from other chromosomes. Such amplified sequences were mapped in detail by genome-wide array comparative genomic hybridization in ring-containing tumor samples from soft tissue (n=15) and bone (n=6), using tiling resolution microarrays, encompassing 32 433 bacterial artificial chromosome clones. The DNA copy number profiles revealed multiple amplification targets, in many cases highly discontinuous, leading to delineation of large numbers of very small amplicons. A total number of 356 (median size: 0.64 Mb) amplicons were seen in the soft tissue tumors and 90 (median size: 1.19 Mb) in the bone tumors. Notably, more than 40% of all amplicons in both soft tissue and bone tumors were mapped to chromosome 12, and at least one of the previously reported recurrent amplifications in 12q13.3–14.1 and 12q15.1, including SAS and CDK4, and MDM2, respectively, were present in 85% of the soft tissue tumors and in all of the bone tumors. Although chromosome 12 was the only chromosome displaying recurrent amplification in the bone tumors, the soft tissue tumors frequently showed recurrent amplicons mapping to other chromosomes, that is, 1p32, 1q23–24, 3p11–12, 6q24–25 and 20q11–12. Of particular interest, amplicons containing genes involved in the c-jun NH2-terminal kinase/mitogen-activated protein kinase pathway, that is, JUN in 1p32 and MAP3K7IP2 (TAB2) in 6q24–25, were found to be independently amplified in eight of 11 cases with 12q amplification, providing strong support for the notion that aberrant expression of this pathway is an important step in the dedifferentiation of liposarcomas.
Several bone and soft tissue tumor entities are characterized cytogenetically by supernumerary ring chromosomes or giant rod marker chromosomes in karyotypes with few or no other chromosome aberrations (Mitelman et al., 2005). The majority of these tumors are low-grade malignant neoplasms that include well-differentiated liposarcoma/atypical lipomatous tumor (ALT), subgroups of malignant fibrous histiocytoma (MFH), myxofibrosarcoma, parosteal osteosarcoma and dermatofibrosarcoma protuberans (DFSP). The origin of the ring chromosomes, which frequently vary in number and size between cells from the same tumor, cannot be disclosed by chromosome banding techniques owing to the diffuse banding pattern and morphological variability. Fluorescence in situ hybridization (FISH) analyses have revealed that these structures, in most cases, contain material from chromosome 12, apart from in DFSP where chromosome 17 and 22 sequences, including the PDGFB/COL1A1 fusion gene, are present in rings (Dal Cin et al., 1993; Szymanska et al., 1996; Chibon et al., 2002; Sirvent et al., 2003). With few exceptions, it is the central part of the long-arm of chromosome 12 (12q) that is present in multiple copies in the ring chromosomes. The amplifications may be discontinuous and vary in size, but frequently include chromosome bands 12q14 and 12q15 (Berner et al., 1996). The MDM2 gene is always amplified, whereas several other genes, such as SAS, GLI, CDK4 and HMGA2, are frequently coamplified (Meltzer et al., 1991; Nilbert et al., 1994; Pedeutour et al., 1994; Gamberi et al., 2000; Gisselsson et al., 2002).
The composition of the ring and giant marker chromosomes is often quite complex and may include material from two or more chromosomes. Apart from the inclusion of chromosome 12 material, direct evidence through FISH analysis has shown that a variety of chromosomes may be involved, in particular chromosome 1 (Pedeutour et al., 1994; Forus et al., 1995a; Meza-Zepeda et al., 2001; Nilsson et al., 2004). Similarly, indirect evidence has been obtained from chromosome-based comparative genomic hybridization (CGH) (Pedeutour et al., 1999; Chibon et al., 2002; Micci et al., 2002; Coindre et al., 2004). The profiles of genomic imbalances show many similarities between ALT, dedifferentiated liposarcoma (DDLS) and MFH, which all share amplification of 12q14–15 in practically all cases, and in which gain of 1q21–23 is common. However, also some distinct differences have been reported (Chibon et al., 2002). For example, gain of sequences from 1p32, 6q23 and 12q24 were absent or very rare in ALT but fairly common among the other soft tissue tumor types. Available data indicate a considerable biological overlap between low-grade MFH and lipomatous tumors (Chibon et al., 2002; Coindre et al., 2003). Based on chromosome CGH data, the 12q amplicon extends from 12q13 to 12q21 in most cases, with a peak in 12q14 and 12q15, but may occasionally include both more distal and more proximal regions. The 1q amplicon is usually located between 1q21 and 1q25 with a peak incidence in 1q23, whereas the 1p amplicon seems to be narrow and commonly includes only 1p32.
In the present study, soft tissue and bone tumors with ring and/or giant marker chromosomes and few or no other chromosome aberrations were investigated by array CGH. For this purpose we used whole-genome tiling resolution bacterial artificial chromosome (BAC) microarrays, encompassing 32 433 BAC clones. To determine whether any association between patterns of DNA copy number alterations and tumor histotype could be detected, tumors with several different diagnoses were included in the investigation.
The array CGH analysis revealed DNA copy number alterations in all 13 soft tissue tumors and in all six bone tumors (Figure 1a, Table 1). Although several single-copy gains and losses of larger chromosomal segments were observed, the copy number changes were dominated by genomic amplifications. As a lot of these demonstrated highly discontinuous patterns, large numbers of very small amplicons were seen in the majority of cases. In many instances, the amplification levels were higher than fivefold, including extreme cases in which more than 30-fold amplification were observed. Even though individual amplicons were dispersed throughout most of the genome, marked aggregations were observed in specific chromosomes, mainly 1 and 12 (Figures 1 and 2). The tumor recurrences, samples 1b and 11b, showed DNA copy number profiles that were highly similar to the corresponding primary tumors (Figure 1, Table 1).
Altogether, 446 amplicons were identified among the 21 samples, 356 in the soft tissue tumors (2–54 amplicons per sample) and 90 in the bone tumors (4–28 amplicons per sample). The number of chromosomes involved in amplification in each case varied from 1 to 14 (case 9); only chromosome 4 was never involved. Analysis of primary tumor data from all 19 patients on average indicated a higher amplification frequency in the soft tissue tumors than in the bone tumors (23 vs 15 amplicons per case). Moreover, the analysis showed that amplicon sizes were significantly smaller among the former (median size: 0.64 Mb) than among the latter (median size: 1.19 Mb; P<0.01, Mann–Whitney U-test). Neither the number nor the distribution of amplicons distinguished the four soft tissue tumors that metastasized from those that did not, or the high-grade from the low-grade osteosarcomas.
Regarding the genomic localization of the amplicons, chromosome 12 was by far most frequently affected (17 of 19 cases), harboring more than 40% of the amplicons in both tumor groups. In the soft tissue tumors, also chromosomes 1 and 6 were commonly affected, containing 25 and 6% of the amplicons in this group, respectively. Apart from chromosome 12, the only chromosome amplified in more than one bone tumor was chromosome 5. However, this chromosome did not contain any overlapping amplicons. A complete description of all amplicons is available in Supplementary Table 1.
To identify genomic regions likely to contain genes of importance for tumor development, a list of recurrent amplifications was compiled (Table 2). In the soft tissue tumors, segments amplified in 2–11 of 13 cases were identified in 1p, 1q, 3p, 6q, 12p, 12q and 20q. Because of the high amplification occurrence and complex distribution within chromosomes 1 and 12 in this group, amplification frequency plots were generated for these chromosomes (Figure 3a and b). The profile for chromosome 1 showed a distinct peak in 1p32, indicating a recurrently amplified region of 1.18 Mb that contains four annotated genes, including JUN (Table 2). In 1q, three frequently amplified regions, all located within 1q23.2–1q24.3, were detected. The chromosome 12 profile revealed two narrow peaks of amplification in 12q13.3–14.1 and 12q15, respectively, affected in more than 60% of the soft tissue tumors. The proximal 0.42 Mb region contains 17 genes, including SAS and CDK4, and the distal 0.75 Mb region comprises 16 genes, including MDM2. Other, less frequent, recurrent amplifications in the soft tissue tumors were seen in 3p11.1–12.1, 6q24.3–25.1 and 20q11.2–12.1 (Table 2).
The only chromosome showing overlapping amplifications in the bone tumors was chromosome 12. The amplification analysis revealed three small regions in 12p, all amplified in two of six cases (Table 2). The most distal segment, located in 12p13.3, is 0.62 Mb in size and contains seven genes, including CCND2, FGF6 and FGF23. In 12q, the frequency plot clearly showed two major target regions, both amplified in all six cases (Figure 3c). The proximal, in 12q13.3–14.1, is 0.91 Mb in size and covers 17 genes, including SAS and CDK4. The distal 1.02 Mb region, located in 12q15, contains eight genes, including MDM2 (Table 2). Notably, the 12q13.3–14.1 commonly amplified region was highly similar in the soft tissue and bone tumors, whereas the equivalent in 12q15 only overlapped partially. Combining data from all 19 cases, additional recurrently amplified regions were observed in 5p14.1–15.2, 8q13.3, 14q11.2 and 16q22.2–22.3 (Supplementary Table 1).
Genomic losses were few and mostly sporadic. The only recurrent aberration encompassed a large chromosome segment, namely the entire or the major part of 9p (Figure 1a). In case 3, there was loss of one copy of 9p, together with gain of one copy of 9q. In case 4, a heterozygous partial deletion of 9p, from 9p13.3 to 9pter, was found. Finally, case 17 showed loss of one entire copy of chromosome 9.
To validate the array CGH results and to determine the chromosomal organization of a few selected amplicons, a whole-chromosome-painting (wcp) Y probe and BAC clones on chromosomes 3, 6 and 12 were used for metaphase FISH analyses of four primary tumors. Array CGH analysis of an inflammatory MFH (case 10) showed a copy number profile with amplification of 3p as the sole aberration. This amplification was confirmed by FISH, using three BAC clones within a 6 Mb region in 3p11–12. The amplified sequences were localized in giant marker chromosomes in some cells, and in rings in others (Figure 4a). The copy number profile of the second tumor, a DDLS (case 3), indicated amplifications on several chromosomes, including sequences overlapping with the 3p amplicon in case 10 and various segments on 12q (Figure 4b). FISH analysis confirmed a recurrently amplified region in 3p11–12, and demonstrated high-level amplification of a sequence covering the MDM2 gene in 12q15 (Figure 4b). The third tumor, an MFH (case 7), showed several amplification peaks on both chromosomes 6 and 12, according to the array CGH analysis (Figure 4c). Two of the amplicons on these chromosomes, located in 6q24 and 12q15, respectively, were confirmed by FISH analysis (Figure 4c). In the fourth tumor, an osteosarcoma (case 18), a unique amplification of the Y chromosome was identified at array CGH analysis (Figure 1a). This case also showed amplification of several segments in both 12p and 12q. FISH analyses using BAC clones mapped to 12p11 and 12q15, and a wcp Y probe, corroborated the array CGH findings and demonstrated tumor heterogeneity with chromosome 12 sequences amplified in various combinations of rings and/or giant markers in different cells (Figure 4d). The wcp probe confirmed amplification of the Y chromosome in a smaller proportion of the cells, always localized at the ends of marker chromosomes.
In the present study, we used genome-wide tiling-resolution BAC microarrays to characterize DNA copy number alterations in 21 bone and soft tissue tumor samples, containing supernumerary ring and/or giant marker chromosomes. The investigated specimens demonstrated aberrations, ranging from single-copy imbalances of entire chromosomes to high-level microamplifications involving only a few BAC clones (Figures 1 and 2).
Apart from heterozygous loss of material from the short arm of chromosome 9 in two of 13 soft tissue tumors and one of six bone tumors, no recurrent deletions were identified. In two of these cases, the findings were expected from the G-band karyotype. Case 17 showed monosomy 9 and case 3 harbored an i(9)(q10) in the more complex of the two hyperdiploid clones (Table 1). The resulting loss of 9p and gain of 9q in the latter tumor was clearly demonstrated at array CGH analysis. The loss of the major portion of 9p in case 4 was not indicated by the karyotype and might be explained by the presence of a subclone that passed undetected at cytogenetic analysis. Monosomy 9 or partial losses of 9p are common in many tumor types, including MFH of several subtypes (Mairal et al., 1999; Simons et al., 2000; Sabah et al., 2005). The tumor suppressors CDKN2A and CDKN2B in 9p21, and possibly other loci, have been implicated in MFH tumorigenesis. By chromosome-based CGH studies, 9p losses in MFH were found in 10–55% of the cases, included the major portion of distal 9p, rarely only 9p21, and four of nine informative cases showed simultaneous loss of 9p21 and over-representation of MDM2 (Simons et al. (2000) and references therein). Whether the large segmental losses of 9p seen in three of 19 cases in the present study play any pathogenetic role remains unknown.
As expected from the present tumors with few other changes than supernumerary ring chromosomes, the majority of gene copy number alterations consisted of genomic amplifications. In total, 446 amplicons were identified; 356 in the soft tissue tumors and 90 in the bone tumors. The amplicons frequently spanned genomic segments less than 1 Mb in size, and occasionally demonstrated more than 30-fold amplification, corresponding to over 60 gene copies. The number of separate amplicons that could be detected in most tumors by the present tiling resolution BAC array by far exceeded the number of amplicons identified in previous studies using chromosome CGH, and allowed a much more accurate delineation of the amplicons. What may appear as a single amplified unit distinguished at the chromosome band or sub-band level by conventional CGH could thus be shown to consist of several separate amplicons.
Although the amplification patterns in soft tissue and bone tumors overlapped with regard to the frequent involvement of 12q segments, several differences were seen. The amplicons were smaller in the soft tissue tumors, bone tumors generally contained fewer amplicons, amplification of 12p sequences was more common in bone tumors, and whereas amplicons were commonly seen in 1p and 1q in the soft tissue tumors, these chromosome arms were affected in none and one of the bone tumors, respectively. Although the number of cases was small, and many different histotypes were included, these differences suggest that the development of rings and markers is associated with different events or selection in bone and soft tissue tumors.
Amplification of sequences from chromosome 12 was observed over the entire tumor panel, and a less marked clustering of amplicons to chromosome 1 was seen in soft tissue tumors (Figure 1). The two commonly amplified regions on 12q were strikingly similar in soft tissue and bone tumors. A 0.42 Mb amplicon in bands 12q13.3–q14.1, present in nine of the soft tissue tumors, shared proximal borders (RP11-571M6) with a 0.91 Mb amplicon seen in all six bone tumors (Table 2). The 0.42 Mb region that was in common for bone and soft tissue tumors contains several oncogenes, including SAS and CDK4, previously shown to be amplified in bone and soft tissue tumors with or without known ring chromosomes (Meltzer et al., 1991; Nilbert et al., 1994; Pedeutour et al., 1994; Berner et al., 1996; Gamberi et al., 2000; Meza-Zepeda et al., 2001; Gisselsson et al., 2002; Nilsson et al., 2004). The other amplicon on 12q that was present in all six bone tumors, covering a 1.02 Mb region in band 12q15, partially overlapped with a 0.75 Mb amplicon present in 11 soft tissue tumors; these two amplicons share a 95 kb region containing a single candidate target gene – MDM2. Thus, the present study provides strong support for the importance of MDM2 amplification for the development of certain subsets of both bone and soft tissue tumors.
Three separate recurrent amplicons were detected in 1q in the soft tissue tumors. Gain or amplification of 1q sequences have previously been described in a variety of soft tissue tumors, and the existence of at least two separate amplicons in 1q21 and 1q23, respectively, has been suggested (Forus et al., 1995a, 1995b, 1998; Szymanska et al., 1997; Kresse et al., 2005). In the former amplicon, three candidate target genes have been proposed, COAS1–3 (Meza-Zepeda et al., 2002). However, their significance for sarcoma development remains unclear, and it could be noted that they were not part of any recurrent amplicon in the present series of tumors. A 7 Mb region around the APOA2 locus in band 1q23 was recently studied more extensively in a series of 10 soft tissue sarcomas with previously known rearrangements of 1q21–23 (Kresse et al., 2005). The results suggested the presence of an 0.8 Mb core amplicon containing 11 known genes, of which ATF6 and DUSP12 were found to show the highest level of amplification. This 0.8 Mb amplicon partly overlaps with one of the three minimally amplified regions we detected in 1q23–24, that is, the 0.53 Mb amplicon extending from RP11-749M10 to RP11-5K23. However, the overlap is restricted to the segment covered by BAC RP11-5K23, which contains three known genes: FCGR2A, FCGR3A/B and HSPA6. At present, none of these genes has a clear role in tumorigenesis.
The complex and frequent involvement of chromosome 1 in soft tissue tumors is further illustrated by the finding of a recurrent amplicon in its short arm. Also gain of 1p sequences has been described before (Chibon et al., 2002), but our data could delineate a 1.18 Mb region in 1p32, containing only four known genes, of which the JUN oncogene is the most likely target. Chibon et al. (2002) have previously shown by conventional CGH analysis that some 20% of MFH tumors display high-level amplification of 12q14–15 sequences, a feature suggesting a biological relationship with well-differentiated and dedifferentiated liposarcomas. Interestingly, out of 22 such cases, 15 showed concomitant 1p32 (n=9) or 6q23 (n=6) amplification. Further studies of the 6q23 amplicon revealed consistent amplification and overexpression of the MAP3K5 gene (Chibon et al., 2004), encoding a protein that is part of the c-jun NH2-terminal kinase (JNK)—mitogen-activated protein kinase (MAPK) signaling pathway. Based on these results, they speculated that aberrant activation of this pathway could affect tumor cell differentiation, a hypothesis elegantly supported by evidence of restored adipocytic differentiation in an MFH cell line treated with inhibitors of MAP3K5. In this context, it is of particular interest to note that also we found frequent, and mutually exclusive, coamplification of 1p32 and 6q sequences, seen in five and three soft tissue tumors, respectively, with 12q14–15 amplification, and that the candidate target gene in the 1p32 amplicon – JUN – is a downstream effector in the JNK-MAPK pathway. The 2.26 Mb amplicon we detected in 6q24–25 was, however, distal to the one described by Chibon et al. (2004), and did thus not include the MAP3K5 locus. On the other hand, it encompassed another gene in the JNK-MAPK pathway, MAP3K7IP2 (a.k.a. TAB2). Expression of the MAP3K7IP2 protein induces MAPK8 (JNK1), which in turn binds to, and phosphorylates, JUN. In line with the results by Chibon et al. (2002) only one of eight soft tissue tumors with concomitant 12q14–15 amplification and 1p32 or 6q24–25 amplification was diagnosed as a liposarcoma, whereas the three tumors with the 12q amplicon, but not the 1p or 6q amplicons, were classified as liposarcoma (two cases) or desmoid tumor. Taken together, the present results thus provide strong support for the hypothesis presented by Chibon et al. (2004) that aberrant activation of the JNK-MAPK pathway is an important step in the dedifferentiation of liposarcomas with 12q amplification.
The finding of an isolated 6 Mb amplification in chromosome bands 3p11–12 in case 10, an inflammatory MFH of the finger, was an odd exception to the otherwise ubiquitous 1q and/or 12q amplification pattern seen in the remaining soft tissue tumors. Soft tissue sarcomas are quite rare in the distal parts of the extremities, but it could be noted that one case of an entity showing a particular predilection for the hands and feet – myxoinflammatory fibroblastic sarcoma – recently was shown to contain amplified material from unspecified parts of chromosome 3, but not 12, in 2–4 supernumerary ring chromosomes (Mansoor et al., 2004). Thus, it is tempting to speculate that there may exist a distinct subset of inflammatory/myxoinflammatory soft tissue sarcomas with a predilection for the distal parts of the extremities characterized by amplification of genes on chromosome 3 rather than on chromosome 12.
Previous studies of gene amplifications have clearly demonstrated that far from all detected gene amplifications in tumor cells are of pathogenetic significance, as, for instance, many amplified genes are not overexpressed (Platzer et al., 2002; Heidenblad et al., 2005). Serendipitous gene amplifications may be particularly common in ring-containing tumors, as the mitotically unstable ring chromosomes often transform into rod-shaped chromosomes, typically capped in both ends by telomeres and subtelomeric sequences from other chromosomes. One good example of this phenomenon was seen in an osteosarcoma (case 18) with amplification of material from chromosome 12, as well as the Y-chromosome. When analysed by metaphase FISH, however, it could be demonstrated that the Y-chromosome material was never present in the ring chromosomes, but always located at the ends of the clonally occurring giant marker chromosomes. Thus, there is reason to be cautious when interpreting the significance of amplified regions from distal parts of chromosomes in ring-containing tumors.
The mechanisms behind ring chromosome formation remain unknown. Typically, bone and soft tissue tumors with supernumerary ring chromosomes harbor two normal chromosomes 12, in spite of the fact that the rings, with few exceptions, contain chromosome 12 sequences. We have previously demonstrated that heterodisomy is retained for those parts of chromosome 12 that are not included in the rings, suggesting that the ring formation occurs owing to errors in the G2 phase of the cell cycle (Mertens et al., 2004). However, models previously proposed for the formation of gene amplification in the form of homogeneously staining regions or double minutes are difficult to apply in the case of ring chromosomes, which, as shown in the present study, are composed of large genomic regions, often from several different chromosomes. A further problem when studying ring chromosomes is that they are mitotically unstable, presumably leading to a strong selection against sequences that do not add proliferative advantage. Thus, the ring chromosomes that are present at tumor diagnosis may be very different from those initially formed. Nevertheless, the consistent presence of material from 12q strongly suggests that the initiating DNA breakage occurs here. In this context it is of interest to note that the frequent 12q13–14 amplicon, containing SAS and CDK4, had a very sharp proximal border, delineated by BAC RP11-571M6 in eight cases and by BAC RP11-746D11 in five cases. This observation, and the fact that the DDIT3 (a.k.a. CHOP) gene, which is rearranged in the pathognomonic t(12;16) in myxoid liposarcomas, is located in RP11-746D11, indicate that sequences in this region are particularly susceptible to breakage, possibly due to recombination-prone sequences in the vicinity of DDIT3 (Kanoe et al., 1999). However, the sharp border could also imply that genes whose overexpression would counteract tumorigenesis are positioned just proximal to the amplicon. This hypothesis is supported by the finding that none of the 20 tumors showed amplification of a 1.5 Mb segment centromeric to the sharp border, and that this segment indeed contains potentially growth-suppressing genes, for example, INHBC and NAB2. Another recurrent amplicon, in 1p32, partially includes the very large DAB1 gene, which was recently shown to map to the FRA1B common fragile site (Smith et al., 2005). Even though none of the five tumors containing this amplicon showed identical breakpoints, they all displayed distal amplification borders within DAB1, again exemplifying how high-resolution array CGH may act as a tool, not only to allow precise mapping of very small imbalances but also to pinpoint genomic segments prone to DNA breakage. Future use of tiling resolution BAC microarrays and even higher resolution oligonucleotide-based arrays will undoubtedly provide new insights as to how and why these chromosomal rearrangements occur.
Materials and methods
Tumor and reference samples
Based on the presence of ring and/or marker chromosomes at previous G-banding analysis, six bone and 15 deep-seated soft tissue tumor samples from 19 patients were selected for array CGH analysis (Table 1). From two patients (cases 1 and 11), a local recurrence and a distant metastasis obtained four and 2 years, respectively, after the primary tumors, were included. The patients consisted of 14 men and five women, 7–80 years old at the time of diagnosis. As a control for normal gene copy number in the array CGH experiments, a DNA pool derived from multiple healthy male donors (Promega, Madison, WI, USA) was used.
32k BAC microarrays
The 32k microarrays were constructed from purified degenerate oligonucleotide-primed polymerase chain reaction products (Jönsson et al., 2005), produced from 32 433 individual BAC clones, processed and purified at the BACPAC Resources center (Oakland, CA, USA). A complete list of the BAC clones, together with genomic position and other detailed information, is available at http://bacpac.chori.org, including direct links to the UCSC human genome browser. For the mapping presented herein, the BACPAC May 2004 data (hg17) were used.
DNA isolation, labeling and microarray hybridization
Genomic DNA was extracted from archived freshly frozen tumor biopsies. After brief thawing, soft tissue tumors were disaggregated through scalpel mincing, and bone tumors using a dismembrator (Mikrodismembrator II, B.Braun, Melsungen, Germany). DNA was isolated using the DNeasy Tissue Kit (Qiagen, Valencia, CA, USA), including the optional RNaseH treatment.
Labeling of test and reference DNA was performed essentially as described previously (Jönsson et al., 2005). In brief, 1.0 μg of tumor and reference DNA was fluorescently labeled with Cy3-dCTP and Cy5-dCTP (Amersham Biosciences, Uppsala, Sweden), respectively, using the Array CGH labeling kit (Invitrogen, Carlsbad, CA, USA), and purified using filter-based spin columns (Cyscribe GFX Purification kit, Amersham Biosciences). Differentially labeled DNA was pooled, mixed with 100 μg Human Cot-1 DNA (Invitrogen), and lyophilized before resuspension in 57 μl hybridization solution (50% formamide, 10% dextran sulfate, 2 × standard sodium citrate (SSC), 2% sodium dodecyl sulfate (SDS), 10 μg/μl yeast tRNA). Probes were heated at 70°C for 15 min and at 37°C for 30 min before hybridization to microarrays for 48–72 h at 37°C. Before hybridization, microarrays were UV-crosslinked at 500 mJ/cm2 and pretreated using the Universal Microarray Hybridization Kit (Corning, Acton, MA, USA) according to the manufacturer's instructions. The slides were washed in 2 × SSC, 0.1% SDS for 15 min, followed by 2 × SSC, 50% formamide (pH 7.0) for 15 min at 45°C, 2 × SSC, 0.1% SDS for 30 min at 45°C, and 0.2 × SSC for 15 min at room temperature. Fluorescence was recorded using an Agilent G2565AA microarray scanner (Agilent Technologies, Palo Alto, CA, USA).
Image and data analysis
Primary data were collected using the GenePix Pro 4.0 software (Axon Instruments Inc., Foster City, CA, USA), and raw result files were deposited into the web-based database BioArray Software Environment (BASE) (Saal et al., 2002). Spots were background-corrected using the median foreground minus the median background signal intensities for both dyes, and log2 ratios were calculated. Unreliable features, marked in the feature extraction software, and spots not showing signal-to-noise ratios ⩾3, for both channels, were removed. Data normalization was performed per array subgrid using the Lowess curve fitting (Yang et al., 2002) with a smoothing factor of 0.33, excluding BAC clones on chromosomes X and Y during the estimation of the normalization function. In the data interpretation, amplicons were defined as regions with at least two consecutive BAC clones showing log2 ratios ⩾1.0, and amplicon sizes given as the longest distance between the two outermost amplified BAC clones. In uncertain regions, showing highly discontinuous amplification, amplicons were considered ended when separated by at least two BAC clones associated with log2 ratios below 0.5. For the amplification frequency plots, a BASE implementation of CGH-Plotter (Autio et al., 2003) was used to define each BAC clone as amplified (numerical value 1) or unamplified (numerical value 0), using an amplification log2 ratio threshold of ⩾0.99, and a moving mean sliding window of three clones. Using the values given from CGH-Plotter, amplification frequencies for BAC clones along frequently affected chromosomes were calculated. When indicated, gene copy number profiles are presented as a moving average (symmetrical two nearest neighbors). Primary data are available upon publication at ArrayExpress (http://www.ebi.ac.uk/arrayexpress).
The probes used for FISH analysis consisted of a commercially available wcp probe specific for the Y chromosome (Vysis, Downers Grove, IL, USA), and BAC clones spanning amplified regions; RP11-80H24 (3p12.1), RP11-81P15 (3p12.1–3p11.2), RP11-91A15 (3p11.1), RP11-307P5 (6q24.3), RP11-1137N1 (12q15), and RP11-564A17 (12p11.2) (BACPAC Resources center). Isolated BAC DNA was labeled with biotin-, fluorescein isothiocyanate-, or Cy3-conjugated dUTP by random hexamer priming (Megaprime DNA labeling system, Amersham, Buckinghamshire, UK). Biotin-labeled probes were detected using DEAC-conjugated avidin. FISH analysis was performed as previously described (Dahlén et al., 2003).
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This work was supported by the Swedish Cancer Society, the Swedish Children's Cancer Foundation, the Crafoord Foundation, the Gunnar Nilsson's Cancer Foundation, the Knut and Alice Wallenberg foundation via the Swegene program, the Ingabritt and Arne Lundberg Foundation, and the Royal Physiographic Society in Lund.
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Heidenblad, M., Hallor, K., Staaf, J. et al. Genomic profiling of bone and soft tissue tumors with supernumerary ring chromosomes using tiling resolution bacterial artificial chromosome microarrays. Oncogene 25, 7106–7116 (2006). https://doi.org/10.1038/sj.onc.1209693
- array-based CGH
- gene amplification
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