Chromosomal imbalances detected in primary bone tumors by comparative genomic hybridization and interphase fluorescence in situ hybridization

We applied a combination of comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH), to characterize the genetic aberrations in three osteosarcomas (OS) and one Ewing’s sarcoma. CGH identified recurrent chromosomal losses at 10p14-pter and gains at 8q22.3-24.1 in OS. Interphase FISH allowed to confirm 8q gain in two cases. A high amplification level of 11q12-qter was detected in one OS. The Ewing’s sarcoma showed gain at 1p32-36.1 as the sole chromosome alteration. These studies demonstrate the value of molecular cytogenetic methods in the characterization of recurrent genomic alterations in bone tumor tissue.


Introduction
Recent technological advances in molecular genetics and cytogenetics have made it easier to identify some of the critical events associated with the development of cancer.Specific chromosome aberrations have been identified in some neoplastic diseases.The most remarkable progress has been achieved in hematological cancers.Knowledge concerning solid tumors is less advanced, which includes bone tumors.The Catalog of Chromosome Aberrations in Cancer (Mitelman et al., 2003) presently contains more than 42,600 karyotypically abnormal neoplasms.Osteosarcomas and Ewing's sarcomas account for less than 1% of these cases.
Ewing's sarcoma (ES) is the most frequent bone tumor in children under 10 years of age and the third most common primary malignant bone tumor in adults.Its characteristic alteration is t(11;22)(q24;q12), which, in association with variant translocations involving 11q24 or 22q12, is described in 90% of the cases.Trisomies 8 (44%) and 12, and der(1;16) leading to trisomy 1q have been described as additional changes.(Armengol et al., 1997).Thus, gains are much more frequent than losses.Armengol et al. (1997) studied Ewing's tumors by CGH and detected high-level amplifications in the 8q13-q24 and 1q regions, and minimal gains of the whole chromosomes 8 and 12, and 1q.Chromosomal aberrations acquired in addition to the ES rearrangements have been postulated to serve as markers for a more refractory disease (Kullendorff et al., 1999), specifically, gains in chromosomes 1q (Armengol et al., 1997), 8, and 12 (Maurici et al., 1998, Zielenska et al., 2001a), and deletions of the short arm of chromosome 1 (Hattinger et al., 2002).Other authors have shown that gains in the long arm of chromosome 1 resulting from an unbalanced translocation t(1;16) have a negative impact on survival (Hattinger et al., 1999(Hattinger et al., , 2002;;Brisset et al., 2001).Polyploidy is also thought to be of prognostic value for predicting progressive disease or local recurrence, and karyotypes with additional structural chromosomal alterations are also considered to be associated with an unfavorable outcome (Kullendorff et al., 1999).
We studied four pediatric bone tumors (three osteosarcomas and one Ewing's sarcoma), not exposed to chemotherapy prior to the analysis, by CGH.To confirm or rule out chromosomal gains, FISH using 8q23 specific probes was used in two of the OS cases.

Patients and tumor specimens
CGH was performed on three osteosarcomas and one Ewing's sarcoma obtained from the Department of Orthopedics (FMRP, USP-Ribeirão Preto, SP, Brazil) (Table 1).Immediately after surgery, the tumor samples were frozen at -80 °C until extraction of high-molecular-weight DNA.Each sample was histopathologically evaluated to ensure the presence of at least 80% of tumor cells.Histopatho-logical classification was performed according to the WHO International Classification of Tumors (1990).None of the patients had undergone any kind of therapy prior to specimen collection.DNA was prepared using standard methodology with proteinase K, extracted by phenol-chloroform, and precipitated with 100% ethanol.DNA samples were stored at -20 °C.Informed consent was obtained from all the patients prior to sampling.

Comparative Genomic Hybridization (CGH)
Metaphase spreads from normal lymphocytes were prepared using standard protocols.The slides were aged for 3-4 days prior to denaturation at 72 °C in 70% formamide/2XSSC, followed by dehydration in an ethanol series.Prior to hybridization, the slides were treated with 0.1 µg/mL proteinase K in 20 mM Tris pH7.5, 2mM CaCl 2 .CGH was performed according to a standard protocol (Kallioniemi et al., 1992) with some previously described modifications (Ojopi et al., 2001), using a QuipXL Genetics Workstation (Vysis, Downers Grove, IL, USA).The cut-off values for the fluorescence intensity ratio were set at 1.20 and 0.80, with a 95% confidence limit.Mean ratio profiles were determined from the analysis of 9 to 12 metaphase cells.Gene amplification was defined as gain ratios ≥ 1.5.The following regions were excluded from analysis: centromeres, acrocentric p-arms, telomeres, and heterochromatin-rich areas (Kallioniemi et al., 1994;Ojopi et al., 2001).In addition, "reverse labeling" by exchanging biotin and digoxigenin labeling of tumor and normal DNA was used.

Fluorescence in situ hybridization (FISH)
A tissue fragment from each of two OS samples was cultured for 5-8 days and processed for cytogenetic analysis, according to previously described procedures (Dracopoli, 2002).Control slides were made from phytohemagglutinin-stimulated normal male lymphoblasts.Sequences BAC RP1179E1 (ATCC #59904, Rockville, MD) and chromosome 8 centromere PMJ 128 were used as probes.The DNA probe BAC RP1179E1 was labeled with biotin-14-dATP (Gibco BRL) and the probe PMJ 128 was labeled with digoxigenin-11-dUTP (Gibco BRL).Probe labeling, hybridization, suppression hybridization, detection, fluorescence microscopy and microphotography were performed as previously described (Rogatto et al., 1999).A minimum of 100 cells was evalu-108 Baruffi et al.

Cytogenetics
Clinical findings of the tumors are listed in Table 1.Chromosomal imbalances are summarized in Table 2, and Figure 1 illustrates the composite karyogram.Gains predominated over losses for all chromosomes.High copy-number amplification was observed at 11q12-qter in one OS sample (FMRP3).The three OS tumors had a common region of gain on chromosome 8 (8q22.3-24.1),although in two of them (cases FMRP2 and FMRP3) these gains were not significant at the 95% confidence limit.The most common losses were at 10p14-pter in two OS.The CGH results in ES revealed gain involving 1p32-36.1.The characteristic t(11;22) was not detected in the ES tumor by RT-PCR methods (data not shown).
Interphase FISH analysis of chromosome 8 centromere and 8q23.2 Since chromosome 8 was found to be the commonly gained chromosome in the OS samples, a more extensive analysis of this numerical change was performed by interphase FISH in cases FMRP2 and FMRP4.In

Discussion
Comparative genomic hybridization is a powerful genomic screening technique and has been widely used to analyze various tumors, including OS and ES.We used CGH and interphase FISH for direct analysis of three OS and one ES.The pattern of genomic alteration observed was consistent with previously published CGH data.The most frequent changes in OS were gains of 8q22, losses of 10p14-pter, and a high level of amplification at 11q12-qter.
Chromosome 8 has previously been found to show copy number changes, rearrangements, and amplifications in OS (Fletcher et al., 1994;Hoogerwerf et al., 1994;Bayani et al., 2003), as well as in many other cancers (Knuutila et al., 1998, Mitelman et al., 2003, Veiga et al., 2003).Previous CGH analyses in OS have revealed the gain and/or amplification of 8q and loss of 8p (Forus et al., 1995;Tarkkanen et al., 1995;Szymanska et al., 1996;Brinkschmidt et al., 1998;Tarkkanen et al., 1998Tarkkanen et al., , 1999;;Mertens et al., 2000;Stock et al., 2000;Zielenska et al., 2001b).In this study, chromosome 8 was found to be the most frequently involved in chromosomal aberrations.Three tumors had a common region of gain at 8q22.3-24.1.Using interphase FISH, we demonstrated the presence of CGH and bone tumors 109 *not significant at 95% confidence limit.this amplification in two samples, although, by CGH, one of them showed a non-significant gain of 8q2.The combined approach of CGH and interphase FISH has greatly increased the ability to detect gains within the 8q2 region in OS.Tarkkanen et al. (1999) reported that patients with tumors displaying a copy number increase at 8q21.3-22, 8qcen-q13, and/or 1q21 are believed to show a trend toward short overall survival.In contrast, Stock et al. (2000) observed that, in 16 OS cases, copy number increases at 8q and 1q21 did not have an unfavorable impact on prognosis.Amplification of 8q, particularly involving the CMYC gene and its surrounding region at 8q24.1, has been described in breast, prostate, lung, colon, head and neck, gastric, acute myeloid leukemia, and brain tumors (Nesbit et al., 1999).In osteosarcoma, this genetic alteration has been described at variable frequencies (Ladanyi et al., 1993, Ueda et al., 1998, Gamberi et al., 1998).In a study on 27 OS, Ladanyi et al. (1993) found 7% of CMYC amplification.In another study, CMYC expression appeared elevated in 9/21 (42%) patients who relapsed, and in 4/17 (23%) patients who remained disease-free (Gamberi et al., 1998).Recently, using FISH and CGH, Stock et al. (2000) demonstrated the presence of two distinct 8q loci involved in the pathogenesis of OS, one located more proximally at 8q21, the other more distally at 8q23-24.1.Three tumor samples displayed only  gain of 8q23 sequences, without involvement of the CMYC oncogene, as verified by FISH (Stock et al., 2000).Tsuneizumi et al. (2001) described an overrepresentation of the EBAG9 gene, CMYC, or both, in 58/129 breast tumors.The EBG9 gene was increased exclusively in 16/27 tumors showing gains at 8q23.This estrogen-responsive gene was found to be identical to RCSA1, a cancer cell-surface antigen implicated in immune escape.We used the RP11-79E1 genomic probe for FISH analysis, mapped at 8q23, where the EBAG9 gene is located.Since 8q22.3-24.1 is the minimal commonly amplified region in OS, there are at least three potential candidate genes: CMYC, TNFRSF11B (this protein is an osteoblast-secreted decoy receptor that functions as a negative regulator of bone re-absorption), and EBAG9.However, this region is large, and it is hard to determine which specific gene(s) could confer a selective driving force, as copy number increases are acquired during OS tumorigenesis.
Amplification of chromosome band 11q12-qter has been found in OS previously (Stock et al., 2000;Zielenska et al., 2001b).Gene amplification is one of the pathways leading to activation of protooncogenes, which may contribute to tumor progression.The identification of amplified genes in tumor cells thus contributes to the understanding of the neoplastic process.We detected DNA amplification of 11q12-qter in OS case.This region includes the cyclin D1 gene, mapped to 11q13.Maelandsmo et al. (1995) found high CCND1 expression by Northern analysis in 22% of osteosarcomas, and cyclin D1 amplification in 4%.Wei et al. (1998) found elevated cyclin D1 expression by immunohistochemistry in only 4% of cases.In addition, in head and neck tumors, amplification of this region has been associated with a higher metastatic potential (Muller et al., 1994).Case FMRP3 with amplification of 11q in addition to gain at 8q11.2-22.3 and 1p13-21, had a poor survival; the patient died of the disease, after 2 years with metastasis.Further studies on these specific high-level amplification regions are necessary to identify the oncogenes which play crucial roles in tumor initiation and/or tumor progression of different OS subsets.
In our study, two OS cases had losses involving 10p14-pter.Recently, Baruffi et al. (2002) reported deletions involving 10p13-pter in three cases of giant-cell bone lesions.There are 66 genes or hypothetical genes mapped to this region (http://cgap.nci.nih.gov/Genes/GeneFinder),including DNMT2 (DNA cytosine-5-methyltransferase 2), located at 10p15.1.Recently, Schultz et al. (2002) found an association between DNA hypomethylation and chromosome 8 aberrations (loss at 8p and gain at 8q21 and 8q23-qter) in prostate cancer.The chromosomal alteration and DNA hypomethylation tended to be frequent in higher-stage tumors and associated with the presence of metastases.The authors suggest that hypomethylation facilitates this particular chromosomal change.Similarly, it is conceivable that the loss of the DNMT2 gene at 10p is re-lated to hypomethylation, and, in association with gain at 8q, contributes to progression of the OS.
Ewing's sarcoma is the most frequent bone tumor in children under 10 years of age, and the third most common primary malignant bone tumor in adults.The case studied by us was re-evaluated by two pathologists and classified as belonging to the Ewing family of tumors based on histology and staining for the MIC2 gene product.Reverse transcriptase-polymerase chain reaction (RT-PCR) indicated that this tumor was negative for the presence of the ES-specific chromosomal abnormality t(11;22)(q24;q12).It is thus possible that it underwent a variant translocation of the EWS gene, leading to fusion transcripts with ES family members such as ERG on chromosome 21q22, or ETV1 at 7p22, or E1AF at 17q12, or FEV at 2q23.Other chromosomal abnormalities, without the specificity of the primary change, have repeatedly been detected in ES (reviewed in Sandberg and Bridge, 2000).In the majority of ES cases described, a net imbalance involving gain of 1q with simultaneous loss of 16q is reported (Sandberg and Bridge, 2000;Mitelman et al., 2003).However, we detected gain at 1p32-p36.1 as the sole chromosomal abnormality in an ES from a one-year-old patient.Loss of 1p36 has been associated with poor outcome in Ewing's sarcoma (Hattinger et al., 1999).Obviously, the small sample size in the present study does not allow a conclusion to be drawn.
Further studies are required increasing the sample size and evaluating the exact extension of the aberrations by FISH with locus-specific probes in order to define the genes involved in gains and losses detected by CGH, in particular losses at 10p and gains at 8q in OS.

Figure 1 -
Figure 1 -Graphic representation of the CGH data obtained from three osteosarcomas and one Ewing's sarcoma.Bars on the right side of each chromosome denote gain, and on the left side loss of a sequence: (A) FMRP2, (B) FMRP3, (C) FMRP4, (D) FMRP9.(E) Interphase FISH in two OS (FMRP2 and FMRP4) confirming the presence of extra chromosome 8q23.2sequence (green signals).The arrows indicate two signals for chromosome 8 centromere (red signals).

Table 1 -
Clinical data on three osteosarcoma (OS) and one Ewing's sarcoma (ES) cases.
Table 3 the proportion of nuclei exhibiting additional signals is in-dicated as a percentage of the total number of nuclei scored.Examples of the interphase FISH analysis are shown in Figure 1.