GENES, CHROMOSOMES & CANCER 48:468–479 (2009)

Genomic Instability in Giant Cell Tumor of Bone.A Study of 52 Cases Using DNA Ploidy, RelocalizationFISH, and Array-CGH Analysis

Linda Moskovszky,1 Karoly Szuhai,2 Tibor Krenacs,1 Pancras C. W. Hogendoorn,3 Miklos Szendro†i,4

Maria Serena Benassi,5 Laszlo Kopper,1 Tibor Fule,1 and Zoltan Sapi1*

11st Departmentof Pathology and Experimental Cancer Research,Semmelweis University,�llo†iu¤ t 26,Budapest1085,Hungary2Departmentof Molecular Cell Biology,Leiden University Medical Center,Albinusdreef 2,Leiden 2300 RC,The Netherlands3Departmentof Pathology,Leiden University Medical Center,Albinusdreef 2,Leiden 2300 RC,The Netherlands4Departmentof Orthopaedics,Semmelweis University,Karolina u¤ t 27,Budapest1113,Hungary5Istituti Ortopedici Rizzoli,Laboratoryof Oncologic Research,Via di Barbiano1/10,Bologna 40136,Italy

Genetic instability in relation to clinical behavior was studied in 52 cases of giant cell tumor of bone (GCTB). Ploidy was

determined in the mononuclear cell population by using native cell smears and image cytometry. A relocalization technique

allowed fluorescent in situ hybridization (FISH) analysis of CD68-negative neoplastic cells for numerical changes of chromo-

somes X, 3, 4, 6, 11, and telomeric association on 11p. Genome-wide alterations were tested using array comparative

genomic hybridization (array-CGH) on magnetically separated CD68-negative tumor cells. CTNNB1, TP53, and BCL2 pro-

tein expression was also analyzed in formol-paraffin sections to see if their pathways are involved in the development of

chromosomal instability. CD68-positive histiocytes showed no significant numerical chromosome and telomeric alterations.

Based on ploidy values and clinical outcome, we could distinguish five groups as follows: diploid nonrecurrent (n ¼ 20),

tetraploid nonrecurrent (n ¼ 6), diploid recurrent (n ¼ 5), tetraploid and/or aneuploid recurrent (n ¼ 14), and malignant

cases (n ¼ 7). Random individual-cell aneusomy was significantly (P < 0.001) more frequent in the recurrent groups

(36.01 � 11.94%) than in the benign nonrecurrent cases (10.65 � 3.66%). The diploid recurrent group showed significantly

(P < 0.001) increased balanced aneusomy compared with the diploid nonrecurrent group and the tetraploid nonrecurrent

group represented eusomic polysomy. Array-CGH and FISH showed clonal aberrations almost exclusively in the malignant

group. None of the protein markers tested showed significant correlation with elevated aneuploidy/polysomy (P ¼ 0.56).

Our results show that ploidy determination combined with FISH analysis may help predicting recurrence potential of

GCTB and suggest that chromosomal abnormalities superimposed on telomeric associations could be responsible for an

aggressive clinical course. VVC 2009 Wiley-Liss, Inc.

INTRODUCTION

Giant cell tumors of bone (GCTB) predomi-

nantly occur in the third and fourth decades of life

as osteolytic neoplasms of potentially aggressive

growth (Varanasi et al., 1999). Histologically, the

tumor is composed of sheets of mononuclear cells

admixed with numerous osteoclast-like giant cells.

Although about 80% of GCTB have a benign

course, local recurrences (20–50%), malignant

transformation at the time of recurrence (10%),

and pulmonary metastases (1–4%) may occur, even

in cases with previously benign histology (Szen-

dro†i, 2004). The most significant factor influencing

recurrence rate is the type of surgical intervention

(en-block resection versus curettage with graft)

and the local use of adjuvants (ethanol, phenol, or

bone cement) (Lausten et al., 1996; Labs et al.,

2001; Szendro†i et al., 2003). To reduce radical sur-

gical interventions, factors that predict GCTB pro-

gression are needed. To date, there have been

no clinical, radiographic, biological, or histological

characteristics revealed to predict the clinical

course of GCTB. It is agreed that neither the

grading system proposed by Jaffe and Dahlin

based on histological features (e.g., the number of

mitoses, vascular invasion) nor the consideration of

other clinicopathological parameters such as age,

gender, tumor size, anatomical site, presence of

Additional Supporting Information may be found in the onlineversion of this article.

Supported by: KP-6 European Commission-granted Network ofExcellence, Grant number: G.0117.06.

*Correspondence to: Zoltan Sapi, Semmelweis University, 1stDepartment of Pathology and Experimental Cancer Research,Ulloi ut 26, 1085 Budapest, Hungary. E-mail: zsapi@freemail.hu

Received 8 November 2008; Accepted 4 February 2009

DOI 10.1002/gcc.20656

Published online 25 February 2009 inWiley InterScience (www.interscience.wiley.com).

VVC 2009 Wiley-Liss, Inc.

pathological fracture at diagnosis, cortical involve-

ment, and distance from the joint, allow prediction

of the clinical course of GCTB (Sanerkin, 1980;

Masui et al., 1998a; Dominkus et al., 2006). The

prognostic significance of stratification (Enneking,

1983; Campanacci et al., 1987) based on clinical,

radiographic, and histological appearance and that

of DNA cytometry have also been disputed (Man-

kin et al., 1985; Ladanyi et al., 1989; Sara et al.,

1990; Siebenrock et al., 1998; Szendro†i et al.,

2003). Furthermore, the expression of none of the

proteins studied in GCTB, including those poten-

tially involved in cell cycle regulation/oncogenesis

(TP53, CDKN1B, CCND1, MYC, TGFBR2)

(Gamberi et al., 1998; Masui et al., 1998b; Kauz-

man et al., 2003; Kawaguchi et al., 2004; Kandel

et al., 2006), the stimulation of osteoclast-differen-

tiation and angiogenesis (VEGF) (Kumta et al.,

2003; Knowles and Athanasou, 2008), bone resorp-

tion (IL6) (Gamberi et al., 2004), or in the degrada-

tion of extracellular matrix (MMP9, PLAU)

(Kumta et al., 2003; Gamberi et al., 2004), have

been found to be of prognostic relevance in a sig-

nificant number of GCTB cases.

Although giant cells play an important role in

the osteolysis, they have been shown to be reac-

tive blood-derived osteoclasts (Forsyth et al.,

2009) and the mononuclear cells of stromal phe-

notype are considered the neoplastic population

(Joyner et al., 1992). However, in GCTB, the

neoplastic CD68-negative cells are mixed with

CD68-positive reactive histiocytes. Most of the

published reports have analyzed this mixed cell

population without separation, which may chal-

lenge the results. Therefore, in this study, we set

up a relocalization technique allowing to examine

genetic instability exclusively in the neoplastic

cells. Purified CD68-negative cells were used to

screen for numerical changes of chromosomes X,

3, 4, 6, 11, and telomeric associations involving

11p by using a multicolor fluorescent in situ

hybridization (FISH) technique and potential

gene losses and gains tested with array compara-

tive genomic hybridization (array-CGH) (Inazawa

et al., 2004). Image cytometry combining ploidy

analysis with the determination of cell prolifera-

tion activity was also performed on the mononu-

clear cell population (Antal et al., 1999, 2000;

Murata et al., 1999). In searching for the potential

cause of the genetic instability, we also tested

the expression of TP53, CTNNB1, and BCL2 in

formol-paraffin sections of the same cases.

MATERIALS AND METHODS

Patient Selection

A total of 52 snap-frozen tissue samples were

selected from the tumor bank of the Semmelweis

University Orthopaedic Clinic (44 cases) and Isti-

tuti Ortopedici Rizzoli Laboratory of Oncologic

Research (8 cases) based on the availability of

complete clinical follow-up including surgical and

radiological reports. There were 25 men and 27

women with a median age of 28.5 years (15–67

years) and a median follow-up of 48 months. The

tested GCTBs were localized to the knee region

(50%), distal forearm (13%), pelvis (10%), and

distal tibia (6%) and included 26 primary benign

nonrecurrent tumors, 17 benign local recurrences,

2 primary benign tumors that recurred, 2 primary

metastasizing cases, 1 metastasis, 1 primary malig-

nant case, and 3 malignant recurrences (Supporting

Table 1). Histological diagnoses were according

to the WHO criteria (Table 1; Figure 1) (Nasci-

mento et al., 1979; Sakkers et al., 1997; Reid et

al., 2002). Of the local recurrences, 11 were first,

6 were second, and 2 were third recurrences. In

TABLE 1. GCTB Classification

Type Features Cases

GCTBa Benign histological appearance: sheets of mononuclear cellsadmixed with numerous osteoclastic giant cells

DNRCb, TNRCc, TRCd, DRCe cases

Metastasizing GCTB Distant metastasis MALf-4, MAL-5, MAL-7Malignancy in GCTB A sarcoma appears at the site of previously documented

GCTB: progression in the same tumorMAL-1, MAL-3, MAL-6

Malignant GCTB/giantcell rich MFH

A high grade sarcoma arises within the GCTB MAL-2

aGCTB, giant cell tumor of bone.bDNRC, diploid non recurrent.cTNRC, tetraploid non recurrent.dTRC, tetraploid recurrent.eDRC, diploid recurrent.fMAL, malignant group.

GENOMIC INSTABILITY IN GIANT CELL TUMOR OF BONE 469

Genes, Chromosomes & Cancer DOI 10.1002/gcc

case of local curettage, all patients were treated

similarly by local adjuvant (phenol 75% and alco-

hol 96%) and bone cement or auto/homologous

bone implantation (Supporting Table 1). In one

case (MAL-4), the patient had been given radia-

tion therapy prior to the genetic analyses.

All cases with a malignant course were inde-

pendently reviewed to avoid inclusion of unrec-

ognized look-a-likes such as osteoclast-rich

osteosarcoma.

All patient information was coded, with com-

plete clinical data available only for physicians

involved in the treatment of these patients.

This study was approved by the ethics commit-

tee of the Semmelweis University (approval num-

ber: 230-151/2006-1018EKU), and all patients

gave their informed consent.

DNA Cytophotometry

DNA cytophotometry was performed on smears

prepared from 58 frozen GCTB samples of the

52 patients. In 4 patients (TRC-7, -8, -10, and

-11) each of the occurrences were analyzed. Fol-

lowing the quantitative determination of DNA

content in tumor cell nuclei after Feulgen stain-

ing (Merck, Darmstadt, Germany), data analysis

was done as previously described (Antal et al.,

1999) by using an image analyzer under the crite-

ria defined by Carey (1994).

DNA-ploidy values were calculated with respect

to a standard reference population (usually lym-

phocytes). Altogether 20–25 lymphocytes and 200

mononuclear cells were measured on each slide

with the exclusion of osteoclast type giant cells.

The given specimen was regarded as diploid if

the DNA-index of cells was between 0.9 and 1.1,

and less than 15% of the nuclei had a 4n DNA

content, representing the G2 fraction. The tumor

was considered aneuploid if the DNA-index was

below 0.9 or between 1.11 and 1.79, and tetra-

ploid if the DNA-index was between 1.8 and 2.2.

The lesion was considered to contain a tetraploid

subpopulation, if beside the diploid peak another

smaller peak (segregated population in the tetra-

ploid range with corresponding G2 phase)

appeared on the histogram.

Regular multiples of the normal chromosomal

sets (4n, 8n, 16n, 32n) are referred to as euploid

polyploidization.

Figure 1. Histological appearances of GCTBs with different bio-logical behavior. (A) Nonrecurrent GCTB with the characteristicmultinuclear osteoclast-like giant cells dispersed in a mixed mononu-clear-cell stroma (case DNRC-2). (B) Primary malignant GCTB. Con-ventional GCTB (upper part of the picture, osteoclasts showed byblack arrows) with abrupt transition to spindle cell tumor; note

atypical mitosis and marked nuclear atypia (inserts) (case MAL-3).(C) Lung metastasis of GCTB (case MAL-8). (D) Secondary malignantGCTB showing atypical spindle cell proliferation with no residualgiant cell tumor (case MAL-4). [Color figure can be viewed in theonline issue, which is available at www.interscience.wiley.com.]

470 MOSKOVSZKY ETAL.

Genes, Chromosomes & Cancer DOI 10.1002/gcc

Immunocytochemistry and Immunohistochemistry

Immunocyto/histochemistry was performed on

either native cell smears or formalin-fixed paraf-

fin-embedded tissue sections by using the iview

DAB detection kit (Ventana, AZ) with indirect

streptavidin-biotin peroxidase complex (S-PO)

method on a Ventana-automated immunostaining

system (Ventana 320). For antigen retrieval of

dewaxed paraffin sections, microwave cooking at

96�C for 30 min in 0.01 M citrate buffer (pH 6.0)

was done. Smears were methanol-fixed before im-

munostaining. All incubations were performed at

37�C with the primary antibodies including

mouse anti-human monoclonal CD68 (1:40, clone

514H12, Serotec, NC), TP53 (1:50, clone DO-7

Dako, Glostrup, Denmark), BCL2 (1:10, clone

124 Dako, Glostrup, Denmark), and CTNNB1

(1:100, clone NCL-B-CAT, Novo-Castra, New-

castle, United Kingdom) for 30 min. Then, the

biotinylated secondary antibody and the S-PO

reagent were sequentially used for 60 min each.

FISH-Relocalization

Five chromosomes were chosen, considering

their size and the combination of available stain-

ing colors, whereas the centromeric and subtelo-

meric regions of the short arm of chromosome 11

were chosen due to its frequent involvement in

telomeric associations (TAS).

After removing the coverslip from the immuno-

stained smears, commercially available probes

were used for each hybridization: centromeric

alpha satellite probes for chromosomes X, 3, 4,

and 6 were simultaneously hybridized. Screening

for possible telomeric alterations was made with

simultaneous hybridization with 11p subtelomeric

and chromosome 11 centromeric probes. All

probes were obtained from Vysis, Abott Molecu-

lar Inc. (Des Plaines, IL).

To distinguish the FISH images of CD68-

positive and negative cells, the Bioview Duet

Automated Scanning System (Bioview, Tel Aviv,

Israel) was used to relocate the previously

scanned CD68 immunostained images and match

with the FISH images of individual cells (Fig. 2).

To determine the specificity of each probe,

FISH was performed on 400 normal control cells

(striated muscle cells and fibroblasts). Based on

the results, cutoff levels for false monosomy and

trisomy (using the mean �2SD) were determined

at 2.93 and 3.54%, respectively (confidence inter-

val: 95%). Next, 100 nonoverlapping, morphologi-

cally well-preserved, mononuclear cells per slide

Figure 2. Relocalization technique. Immunocytochemistry wasdone on smears derived from frozen tissue, using CD68 as a histio-cytic marker. Thereafter, FISH was performed on the same—alreadyscanned—slides hybridizing four centromeric probes simultaneously(X: aqua, 3: red, 4: green, 6: yellow), then on another slide—after

scanning—centromeric and telomeric probes for chromosome 11.With the relocalization technique of Bioview Duet scanning station,we could separate the FISH images of CD68-positive and negativecells. The lower two pictures demonstrate exactly the same areaswith the same position of cells by immunostaining and FISH.

GENOMIC INSTABILITY IN GIANT CELL TUMOR OF BONE 471

Genes, Chromosomes & Cancer DOI 10.1002/gcc

were analyzed, 20–50% of which were CD68-pos-

itive cells depending on the histiocyte-content of

the given sample. Based on the individual centro-

meric signals of the examined chromosomes and

the overall signals, gains and losses were deter-

mined. Analyzing the number of signals of certain

chromosomes separately, the presence of monoso-

mies, trisomies, and other aneusomies were deter-

mined. The resulting frequencies of aneusomies

were calculated for the individual chromosomes as

well as for the individual cells, distinguishing

chromosomal aneusomy, (C-ANEU) and individ-

ual-cell aneusomy (I-ANEU). I-ANEU gives the

combined information on all four chromosomes

(3, 4, 6, and X), whereas C-ANEU represents

numeric alterations for each separate chromosome.

When regular multiple of centromeric signals (4x,

8x) for all tested chromosomes were observed, the

polysomy frequencies were calculated both for

individual chromosomes and for individual cells,

chromosomal polysomies (C-POLY) and individual

cell polysomies (I-POLY), respectively. To define

significant clonal aneusomy, we considered our

and other’s cutoff values (Qian et al., 1996; Yang

et al., 2006) and the Poisson model (Castagne

et al., 2003). Thus, the cutoff values were 20% for

each chromosome. If more than 20% of the CD68-

negative tested cells showed the same gain or loss,

the alteration was considered clonal.

Array-CGH

In GCTB samples, where the percentage of

CD68-negative cells did not reach at least 60% of

total cell content, magnetic cell separation was per-

formed to improve array-CGH analysis. Filtering

the homogenized GCTB cell suspension with

20 lm pore-size Filcon syringe filter (BD Bioscien-

ces, NJ) and incubating it with CD68 antibody-

coated metal beads (Dynabeads M-280 Sheep

anti-Mouse IgG, Invitrogen, Carlsbad, California)

in a ratio of 30 million cells:100 million CD68-

coated beads, the solution was put into a magnetic

field (MagnaRackTM magnetic separation rack,

Invitrogen, Carlsbad, CA), followed by collecting

the CD68-negative cell fraction. DNA was isolated

from frozen tissue as well as from the separated

cell suspension using the High Pure PCR Tem-

plate Preparation Kit (Roche, Mannheim, Germany).

Genome-wide analysis of DNA copy number

changes of samples was performed using slides

containing about 3,500 bacterial artificial chromo-

somes (BACs), obtained from the Welcome Trust

Sanger Institute and produced at the Leiden Uni-

versity Medical Center, the Netherlands. Samples

were labeled and hybridized and analyzed accord-

ing to previously described protocols (Knijnen-

burg et al., 2005, 2007).

Statistics

FISH results were statistically analyzed by

using the Pearson’s v2 test, with results consid-

ered significant at P < 0.001.

RESULTS

Subgroups Based on Ploidy Correlated with

Clinical Features

Out of 26 primary nonrecurrent cases, 20 proved

to be diploid (Fig. 3A) and 6 showed a tetraploid

subpopulation (more than 15% of the measured

cells). Of the 19 recurrent GCTBs, 5 were diploid,

1 was tetraploid, 12 showed a tetraploid subpop-

ulation (Fig. 3B), and 2 cases showed aneuploidy.

Three of the 7 malignant specimens showed a

tetraploid subpopulation, 1 was tetraploid, and the

remaining were aneuploid (Fig. 3C). In 3 out of the

4 examined cases, the ploidy value changed during

its clinical course: from diploid into tetraploid

(TRC-11, Table 2), from diploid into tetraploid sub-

population (TRC-7, Table 2), and tetraploid into

aneuploid (TRC-8, Table 2), respectively. Consid-

ering the relatively small number of patients, the

ploidy-differences found between the various clin-

ical groups were not significant. Further dissection

of results provided us with five different subgroups

clearly separable according to the ploidy value and

the clinical outcome. (1) Diploid nonrecurrent

group (DNRC—n ¼ 20) (we do not expect recur-

rence because of the diploid character); (2) Malig-

nant group (MAL—n ¼ 7) (we expect recurrence

and/or metastasis because of the aneuploid/tetra-

ploid character and/or malignant phenotype); (3)

Tetraploid recurrent group (TRC—n ¼ 14) (we

expect recurrence because of the tetraploid charac-

ter); (4) Diploid recurrent group (DRC—n ¼ 5)

(there is recurrence despite the diploid nature); (5)

Tetraploid nonrecurrent group (TNRC—n ¼ 6)

(there is no recurrence despite the tetraploid na-

ture). (Table 2)

FISH

Control cells

I-ANEU of control cells was 1.54 � 0.58%. C-

ANEUs were 0.00%, 0.97 � 0.35%, 0.33 �0.21%, 0.12 � 0.11%, and 2.87 � 0.96% for

472 MOSKOVSZKY ETAL.

Genes, Chromosomes & Cancer DOI 10.1002/gcc

chromosomes X, 3, 4, 6, and 11, respectively.

The frequency of chromosome 11p subtelomeric

loss was 1.28 � 0.85%.

CD68-positive group

Within the histiocytic cell population, I-ANEU

was 2.59 � 0.55%. There was no significant dif-

ference either between C-ANEUs of CD68-posi-

tive and normal control cells (P ¼ 0.27–0.91) or

between the 11p telomeric alteration frequencies

of these cells.

CD68-negative group

Typical FISH patterns in the different GCTB

groups are demonstrated in Figure 4. I-ANEU

was higher in CD68-negative neoplastic cell pop-

ulation (25.76 � 2.79%) compared with both con-

trol (P < 0.0001) and CD68-positive cell

populations (P < 0.0001) (Fig. 5). Comparing the

C-ANEUs, no significant difference in the fre-

quencies could be shown, which means there was

random aneusomy concerning all four groups

(DNRC, DRC, TNRC, and TRC).

In general, chromosomal gains and losses were

about equal in nonrecurrent and recurrent groups,

whereas gains predominated in the malignant

group (for results concerning C-ANEUs see Sup-

porting Table 2). Polysomy frequency (I-POLY

means) showed increasing tendency among the

different groups (DNRC: 1.13%, DRC: 3.19%,

TNRC: 9.71%, TRC: 11.86%, and MAL: 11.16%)

(Table 2).

The DNRC group showed a low percentage of

aneusomy but it was higher (I-ANEU, 9.45 �0.77%) than in control (P < 0.001) and CD68-

positive cell populations (P < 0.001) (Fig. 5).

The TNRC cases showed slightly higher aneus-

omy (I-ANEU: 14.63 � 3.62%) compared with

DNRC group, but the difference was not signifi-

cant (P ¼ 0.011). This group can be featured by

so-called eusomic polysomy, which means we

found exclusively multiples of 4x or 8x equally

for all of the examined four chromosomes in

fairly high frequency (I-POLY: 9.71 � 3.38%)

(Table 2). In the DRC group, aneusomy (I-

ANEU: 26.06 � 3.30%) was more frequent (P <0.001) compared with the DNRC group, with

gains and losses being about equally common

(3.78% and 3.15%, respectively). The TRC cases

contained a much higher percentage of aneusomy

(39.55 � 4.29%), than the TNRC group (P <0.001). Gains and losses in this group were 5.40%

and 2.89%, respectively. The MAL group showed

a high frequency of aneusomy by FISH (I-

ANEU: 54.02 � 7.24%).

Comparing diploid nonrecurrent to recurrent

and tetraploid nonrecurrent to recurrent groups,

individual-cell aneusomy could be detected more

frequently in recurrent compared with nonrecur-

rent cases (P < 0.001). Analyzing recurrent

GCTBs only, the TRC group contained more

individual-cell aneusomies than DRC cases. Both

I-ANEU and total C-ANEU values were much

higher in MAL compared with nonrecurrent and

recurrent GCTBs (detailed results in Table 2, in

Supporting Table 2 and Fig. 5). No clonal altera-

tion was found in DNRC and TNRC; clonality

could be detected in a few recurrent cases and in

the majority of malignant cases.

Figure 3. Histograms with corresponding DNA indices (DI). (A)Diploid nonrecurrent tumor with one obvious diploid peak (CaseNo: DRC-2). (B) Tetraploid recurrent tumor with the correspondingG2 phase. Two segregated peaks, the smaller is in the tetraploidrange (TNRC-6). (C) Malignant aneuploid case (MAL-3).

GENOMIC INSTABILITY IN GIANT CELL TUMOR OF BONE 473

Genes, Chromosomes & Cancer DOI 10.1002/gcc

TABLE 2. DNA Indices (DI) and I-ANEU Values

CaseDNAIndex I-POLYa (%)

Polysomymean I-ANEUb (%)

Aneusomymean

11p-alteration(%)

11p-alterationmean

DNRCc-1 1.00 0.00 Mean: 1.1352 11.49 Mean: 9.4580 10.00 Mean: 13.8148DNRC-2 0.95 0.00 SDd: 2.2147 3.33 SD: 3.4395 13.64 SD: 4.6958DNRC-3 1.04 0.00 SEMe: 0.4952 8.54 SEM: 0.7691 11.11 SEM: 1.0500DNRC-4 1.05 0.00 Range: 8.2192 8.62 Range: 13.4664 11.11 Range: 20.8827DNRC-5 1.01 1.52 7.58 14.55DNRC-6 1.00 1.98 13.86 11.59DNRC-7 1.04 0.00 12.50 15.56DNRC-8 1.02 0.00 10.87 19.05DNRC-9 0.98 1.16 8.14 23.91DNRC-10 1.00 0.00 4.31 12.22DNRC-11 0.97 0.00 9.91 14.44DNRC-12 1.00 0.00 10.81 8.33DNRC-13 1.00 8.22 12.33 21.11DNRC-14 1.05 6.06 10.61 17.78DNRC-15 0.99 0.00 8.45 16.67DNRC-16 0.96 0.00 2.50 13.33DNRC-17 1.09 0.00 6.94 12.20DNRC-18 1.00 1.79 12.50 3.03DNRC-19 0.95 1.98 9.90 10.00DNRC-20 0.90 0.00 15.97 16.67TNRCf-1 0.96/1.95 2.17 Mean: 9.7154 15.22 Mean: 14.6373 18.48 Mean: 9.3324TNRC-2 1.00/2.02 6.67 SD: 8.2775 20.00 SD: 8.8658 3.85 SD: 4.9740TNRC-3 1.04/2.10 6.82 SEM: 3.3793 9.09 SEM: 3.6195 8.96 SEM:2.0306TNRC-4 0.99/2.10 22.54 Range: 20.3613 5.63 Range: 23.6765 8.85 Range: 14.6321TNRC-5 0.98/2.00 17.24 29.31 6.25TNRC-6 0.96/2.05 2.86 8.57 9.62TRCg-1 1.02/2.08 3.13 Mean: 11.8650 40.63 Mean: 39.5564 7.78 Mean: 14.4713TRC-2 1.05/2.15 22.39 SD: 8.7401 50.75 SD: 16.0575 15.66 SD: 5.7371TRC-3 1.05/2.05 21.88 SEM: 2.3359 26.56 SEM: 4.2915 21.28 SEM:1.5333TRC-4 0.87/1.85 14.10 Range: 31.4286 44.87 Range: 66.4368 17.59 Range: 17.0498TRC-5 1.00/2.00 15.52 6.90 22.76TRC-6 1.01/2.02 12.35 50.62 17.39TRC-7 1.02!1.06/

2.000.00 73.33 13.44

TRC-8 2.1!1.63 31.43 42.86 15.22TRC-9 1.01/2.02 8.70 52.17 11.43TRC-10 1.05/2.02 3.57 35.71 24.83TRC-11 0.96!1.01/

2.00!1.856.32 22.11 7.84

TRC-12 1.05/2.05 8.93 44.64 9.59TRC-13 0.98/2.02 5.17 36.21 9.18TRC-14 0.99/2.00 12.64 26.44 8.60DRCh-1 1.06 1.79 Mean: 3.1914 37.50 Mean: 26.0687 7.49 Mean: 8.2266DRC-2 1.01 2.33 SD: 3.5389 20.93 SD: 7.3933 10.13 SD: 2.2011DRC-3 1.00 9.26 SEM: 1.5827 29.63 SEM: 3.3064 6.38 SEM:0.9844DRC-4 0.91 2.59 Range: 9.2593 21.55 Range: 16.7683 6.16 Range: 4.8145DRC-5 0.90 0.00 20.73 10.98MALi-1 1.06/2.10 27.66 Mean: 11.1586 65.96 Mean: 54.0241 21.43 Mean: 16.1927MAL-2 2.00 17.24 SD: 8.8685 41.38 SD: 19.1594 30.21 SD: 7.3914MAL-3 1.58 6.52 SEM: 3.3520 67.39 SEM: 7.2416 15.12 SEM:2.7937MAL-4 0.92/1.88 12.82 Range: 25.5319 84.62 Range: 50.5728 9.78 Range: 20.4257MAL-5 1.61 5.22 34.78 10.00MAL-6 0.98/2.00 6.52 50.00 15.38MAL-7 1.62 2.13 34.04 11.43

aI-POLY, individual polysomy.bI-ANEU, individual aneusomy.cDNRC, diploid non recurrent.dSD, standard deviation.eSEM, standard error of mean.fTNRC, tetraploid non recurrent.gTRC, tetraploid recurrent.hDRC, diploid recurrent.iMAL, malignant group.

Arrows indicate changes in ploidy-values during clinical course.

11p telomeric alterations

11p deletions that could reflect TAS were pres-

ent in all cases, regardless of ploidy-value or

recurrency-disposition. It appeared slightly but

not significantly more often in malignant cases

(16.19 � 2.8%) compared with the benign

GCTBs (12.80 � 2.17%) (P ¼ 0.15) (Table 2,

Figs. 4K, and 4L).

Array Comparative Genomic Hybridization

Excluding gains and losses involving regions

reported in the DGV database (http://project-

s.tcag.ca/variation/), a normal array-CGH profile

was found in 48/51 tumors.

One recurrent case (TRC-2) showed the imbal-

ances: gain of 4p (3.52–4.4 Mb) with an amplifica-

tion between 4.45 and 4.86 Mb; gain of 12p (152

Kb–3.74 Mb), amplifications in 12q (3.89–3.98

Mb, 5.61–5.78 Mb) (Fig. 6).

Clonal genomic imbalances were observed in 2

out of 7 malignant cases: MAL-2 case: a deletion

of 5q between 8.95–18.00 Mb; MAL-3 case: sev-

eral chromosomal aberrations were seen involving

chromosomal gains: 5, 6, 7, 20, and 21 and losses:

15 and 18. Loss of 1q and 16q was seen. Com-

plex rearrangements partial gains and losses were

observed for several chromosomes: gain of chro-

mosome 2 between 330 Kb and 16.90 Mb, gain

of 4q (7.33–11.18 Mb, 14.39–15.50 Mb, loss of 4q

(17.19–18.26 Mb); loss of 8p (250 Kb–3.00 Mb)

and a gain of 8q (7.72–14.61 Mb); loss of 9p (600

Kb–32.5 Mb); loss of 10p (300 Kb–5.55 Mb) and

gain of 10q (270 Kb–8.95 Mb), loss of 17q from

7.82 Mb to 18p 200 Kb.

CTNNB1 Expression and Chromosomal Instability

Immunohistochemistry was performed in 44

cases. In all positive cases membranous/cytoplasmic

expression of CTNNB1 was seen but no nuclear

positivity could be detected. The membranous/

cytoplasmic positivity was stronger in malignant

than in other cases. Three of the 26 nonrecurrent

(12%), and 8 of the 16 recurrent cases (50%)

showed weak positivity, whereas each of the exam-

ined 2 malignant samples proved to be moderate to

strongly positive. From six of the CTNNB1 posi-

tive cases we made smears, did the CTNNB1

immunocytochemistry and used the same relocali-

zation technique as for separation of CD68-positive

and CD68-negative cells. Here, we were not able

to find any relationship between CTNNB1 positive

cells and aneu/polysomy (P ¼ 0.56).

TP53 and BCL2 Expression

We found no BCL2 positivity in mononuclear

or in multinucleated giant cells, whereas weak

Figure 4. Typical FISH patterns of different GCTB groups. Centro-meric signals of chromosomes X (blue), 3 (red), 4 (green), and 6 (yel-low). (A, B) Disomic cells from male (DNRC-1) and female (DNRC-19) patients from the DNRC group. (C, D) Tetrasomic cells frommale (TNRC-2) (two X signals) and female (TNRC-4) (four X signals)patients. TNRC group featured by tetrasomic cells without aneusomy(eusomic polysomy). (E, F) Male (DRC-1) (chromosome 4-6 disomy,X and 3 monosomy) and female (DRC-4) (chromosome 3-4-X dis-

omy, 6 trisomy) patients from the DRC group. (G, H) Male (TRC-9)(X disomy, 3-4-6 trisomy) and female (TRC-8) (chromosome 3 and Xtetrasomy, chromosome 4 pentasomy and 6 hextasomy) patientsfrom the TRC group. (I, J) Male (MAL-2) and female (MAL-1) patientsfrom MAL group with serious aneusomies. (K, L) Disomic cell with11p telomere loss (DRC-4), and tetrasomic cell with intact telomeres(TNRC-2). Centromeric (red) and subtelomeric (green) signals ofchromosome 11.

GENOMIC INSTABILITY IN GIANT CELL TUMOR OF BONE 475

Genes, Chromosomes & Cancer DOI 10.1002/gcc

(þ) nuclear TP53 positivity in mononuclear cells

was detectable only in 2 malignant cases.

DISCUSSION

Although 80% of GCTBs are benign, local

recurrences and metastases can occur, even in

cases with a previously benign histology. Since

no reliable clinical and/or histopathological

parameters have been identified to predict the

outcome and help reducing radical surgical inter-

ventions in GCTB, we analyzed the genetic

instability in the CD68-negative neoplastic cell

population by using a combination of DNA

cytometry, interphase FISH, and array-CGH in

correlation with the clinical progression of the

disease. We found a significantly elevated num-

ber of random individual-cell aneusomies in

recurrent GCTB compared with nonrecurrent

cases and clonal alterations exclusively in the ma-

lignant group. Eusomic polysomy was seen in

several tetraploid nonrecurrent cases and bal-

anced aneusomy (gains as common as losses) was

more frequent in diploid recurrent than in diploid

nonrecurrent cases. These findings suggest that

chromosomal abnormalities superimposed on telo-

meric associations could be responsible for an

aggressive clinical course in GCTB.

One of the most commonly observed genetic

aberrations in GCTB is TAS (Bridge et al., 1992;

Sciot et al., 2000; Schwartz et al., 2002; Sawyer et

al., 2005). One concept is that telomeric instabil-

ity is responsible for a large degree of intratumor

heterogeneity and serves as a precursor lesion to

subsequent clonal structural aberrations of chro-

mosome 11 in GCTB (Sawyer et al., 2005). How-

ever, recently Forsyth et al. demonstrated that

the degree of telomere shortening in GCTB is

minimal compared with other malignant lesions,

and only the notably shortened telomeres could

be responsible for genetic instability. Telomerase

(hTERT) activity and an inactivated alternative

telomere lengthening mechanism were found,

which suggest that TAS alone does not contrib-

ute to the development of genetic instability

(Forsyth et al., 2008). Considering other genetic

alterations, there are convincing reports on abnor-

mal karyotypes in malignant (metastatic) GCTBs,

Figure 5. (A) I-ANEU values of normal control cells, CD68-posi-tive cell population, and different GCTB groups. Bars representstandard errors of means. Histiocytic cells contain no significant alter-ations compared with normal control cells (P ¼ 0.1992), whereas theneoplastic population shows more aberration compared with histio-cytes (*P < 0.001). Increasing I-ANEU values were seen in the differ-ent GCTB groups, with significant (P < 0.001) differences between

DRC** versus DNRC, TRC*** versus both DRC and TNRC, andMAL**** versus TRC group. (B) C-ANEU values regarding chromo-somes 3 (red), 4 (green), 6 (yellow), 11 (orange), X of females (pink),and X of males (blue) between GCTB groups. Bars represent stand-ard errors of means. [Color figure can be viewed in the online issue,which is available at www.interscience.wiley.com.]

476 MOSKOVSZKY ETAL.

Genes, Chromosomes & Cancer DOI 10.1002/gcc

clearly demonstrating that these tumors differ ge-

netically from the nonmetastasizing ones (Bridge

et al., 1992; Molenaar et al., 1994).

In this study, the DNA content of GCTBs as

measured by image cytometry was diploid in the

majority of cases, but there was a proportion con-

taining tetraploid subpopulations, on the other

hand. Although there was a tendency for recurrent

and malignant tumors to be tetraploid and aneu-

ploid, respectively, the ploidy value by itself was

not able to predict the clinical behavior of GCTB.

For the interpretation of the FISH results, we

set up a relocalization approach and could show

that the CD68-positive mononuclear histiocytes

do not contain chromosomal alterations. Examin-

ing the CD68-negative neoplastic cell population,

we could create five groups according to ploidy

combined with clinical behavior. It was conspicu-

ous, that all five groups showed random individ-

ual-cell aneusomy, i.e., there was no significant

difference between the aneusomy frequencies for

the different chromosomes (for the X chromo-

somes the numbers were corrected for sex) (Fig.

5B). However, the total individual-cell aneusomy

values between groups were significantly differ-

ent (Fig. 5A). Recurrent tumors showed higher

aneusomy rates than nonrecurrent ones, and ma-

lignant cases had higher frequencies than recur-

rent cases (Fig. 5A). The elevated aneusomy rate

in the DRC group (where the rates of mono- and

trisomy were almost equal) compared with the

DNRC group indicates a significantly higher

degree of chromosomal/genomic instability in line

with the tendency of recurrency, as we hypothe-

sized. A high frequency of polysomy was found

in the TNRC group (C-POLY: 9.98–22.54% in

TNRC, whereas 1.61–2.72% in DNRC and

0.00% in CD68-positive histiocytic population),

suggesting eusomic polisomy and euploid poly-

ploidisation, which is a benign phenomenon, e.g.,

in benign schwannomas (Saeter et al., 1988;

Hruska et al., 2004). Thus, tumors in TNRC

group contain tetraploid subpopulations analogous

to some kind of normal tissues (liver, seminal

vesicle) also containing high number of physio-

logical 4n cells (‘‘tetraploid subpopulation’’). This

phenomenon can explain why these tumors do

not recur. The practical conclusion from these

observations is that the increased individual-cell

aneusomy both in diploid and tetraploid tumors

predicts a recurrence, whereas tumors with euso-

mic polysomy are unlikely to recur.

In addition, we performed a whole-genome

screening by using array-CGH analysis (Inazawa et

al., 2004). To exclude bias of results resulting from

inherent tumor heterogeneity, the mononuclear

histiocytic cells and the histiocytic giant cells were

separated from the nonhistiocytic neoplastic cells

by magnetic-bead cell separation. Most previous

studies analyzed mixed mononuclear cell popula-

tions, which might have led to biased results.

Presumably, clonal chromosomal alterations

were found in 5 out of 7 malignant cases with

FISH, and in 2 out of 7 malignant cases with

array-CGH. Thus, clonal alterations could be

detected in 6 out of the 7 malignant cases. This

Figure 6. Array CGH profile after magnetic bead separation. The profile of the CD68-negative (dom-inantly tumor cell) pool showed clear patterns of alterations. Case TRC-2 with high-level amplificationon chromosomes 4 and 12.

GENOMIC INSTABILITY IN GIANT CELL TUMOR OF BONE 477

Genes, Chromosomes & Cancer DOI 10.1002/gcc

supports the conclusion, that a malignant behav-

ior is very likely if a GCTB shows imbalances at

array-CGH or has an aneusomy rate higher than

20% at FISH analysis in alignment to others

(Bridge et al., 1992; Molenaar et al., 1994).

Clonal alterations were found in 3 of the recur-

rent cases by FISH, whereas in only one recur-

rent case by array-CGH (in concordance with

FISH). There were no clonal alterations in the

nonrecurrent cases. The tendency of aneusomy

and clonal alterations suggests that there might

be a biological continuity of genetic instability in

GCTB.

The cause of genetic instability in GCTB still

remains obscure. TAS as causing mechanism is

disputed (Forsyth et al., 2008). To investigate

common factors participating in the regulation of

cell division and associated signaling pathways, we

examined the possible role of CTNNB1, a mem-

ber of the Wnt signaling pathway, as the failure of

this signaling route (usually with the nuclear accu-

mulation of CTNNB1) is well-known to cause

aneusomies and tetrasomies (Yang et al., 2006).

Although we found CTNNB1 positivity in 30% of

our cases, which is in line with a study published

by others on a smaller number of cases (Monaghan

et al., 2001), there was significant correlation nei-

ther between CTNNB1 positivity and the degree

of aneusomy nor between CTNNB1 expression

and the tendency to recur. Nuclear TP53 expres-

sion, indicating possible suppressor gene damage,

was found exclusively in a few malignant cases,

which is also in agreement with earlier studies

(Kandel et al., 2006). However, there might be

TP53 abnormalities which do not cause accumula-

tion of the protein; thus, further studies are needed

to safely exclude TP53 rearrangement as a cause

of genomic instability. As a possible indicator of

apoptotic pathway damage, we investigated the

expression of the anti-apoptotic BCL2, which

was, however, negative in all of our cases.

Although our studied factors behind the genetic

instability were not comprehensive, these and the

negative results of other published works (Gam-

beri et al., 1998; Kauzman et al., 2003; Kumta

et al., 2003; Kawaguchi et al., 2004; Kandel et al.,

2006; Knowles and Athanasou, 2008) support the

concept that genomic instability develops through

other mechanisms in GCTB.

ACKNOWLEDGMENTS

The Departments of Pathology of Semmelweis

University and Leiden University Medical Center

as well as Instituti Ortopedici Rizzoli are partners

of the EuroBoNeT consortium.

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