Frequent deletion of CDKN2A and recurrent coamplification of KIT, PDGFRA, and KDR in fibrosarcoma of...

GENES, CHROMOSOMES & CANCER 49:132–143 (2010)

Frequent Deletion of CDKN2A and RecurrentCoamplification of KIT, PDGFRA, and KDR inFibrosarcoma of Bone—An Array ComparativeGenomic Hybridization Study

Tarja Niini,1* Jose Antonio Lopez-Guerrero,2 Shinsuke Ninomiya,1 Mohamed Guled,1

Claudia Maria Hattinger,3 Francesca Michelacci,3 Tom Bohling,1 Antonio Llombart-Bosch,4

Piero Picci,3 Massimo Serra,3 and Sakari Knuutila1

1Departmentof Pathology,Haartman Institute and HUSLAB,Universityof Helsinki and Helsinki University Central Hospital,Helsinki,Finland2Laboratoryof Molecular Biology,Fundacio¤ n InstitutoValenciano de Oncolog|Ła,Valencia,Spain3Laboratoryof Oncologic Research,Orthopaedic Rizzoli Institute,Bologna,Italy4Departmentof Pathology,UniversitatdeValencia Studi General,Valencia,Spain

Very little is known about the genetics of fibrosarcoma (FS) of bone. We applied array comparative genomic hybridization

(CGH) to identify genes and genomic regions with potential role in the pathogenesis of this tumor. Seventeen patients with

FS of bone were included in the study. Array CGH analysis was carried out in 13 fresh frozen tissue specimens from 11 of

these patients (nine primary tumors and four local recurrences). DNA was extracted and hybridizations were performed on

Agilent 244K CGH oligoarrays. The data were analyzed using Agilent DNA Analytics Software. The number of changes per

patient ranged from 0 to 132 (average ¼ 43). Losses were most commonly detected at 6q, 8p, 9p, 10, 13q, and 20p. CDKN2A

was homozygously deleted in 7/11 patients. Hypermethylation of both p16INK4a and p14ARF was found in 1/14 patients. An

internal deletion of STARD13 was found in a region with common losses at 13q13.1. The most frequent gains were seen at

1q, 4q, 5p, 8q, 12p, 15q, 16q, 17q, 20q, 22q, and Xp. Single recurrent high level amplification was detected at 4q12, including

KIT, PDGFRA, and KDR. No activating mutations were found in any of them. Immunohistochemistry revealed expression of

PDGFRA and/or PDGFRB in 12/17 samples. Moreover, small regions of gains pinpointed genes of particular interest, such as

IGF1R at 15q26.3 and CHD1L at 1q21.1. In conclusion, our analysis provided novel findings that can be exploited when

searching for markers for diagnosis and prognosis, and targets of therapy in this tumor type. VVC 2009 Wiley-Liss, Inc.

INTRODUCTION

Fibrosarcoma (FS) of bone is an extremely rare

neoplasm, which constitutes only about 5% of all

primary malignant bone tumors (Campanacci,

1999). In most cases, patients with FS of bone

are treated with surgery with the possible adjunct

of pre- and/or post-operative chemotherapy regi-

mens, which are very similar or almost identical

to those used for high-grade osteosarcoma (OS).

Right now, on the basis of the available knowl-

edge, there is no reason to differentiate the treat-

ment between these two tumors. The prognosis

of FS of bone is very poor: the 5-year overall sur-

vival being about 34% (Kahn and Vigorita, 2002).

New therapeutic strategies are therefore needed.

A better and more detailed genetic characteriza-

tion of this neoplasm may lead to the identifica-

tion of tumor-related biological markers, which

may become valuable therapeutic targets and

drive the planning of future tailored treatment

regimens.

The diagnosis of FS of bone is very often diffi-

cult, and the differential diagnosis has to be

done, on a histological basis, from malignant fi-

brous histiocytoma (MFH) of bone or from OS.

The immunohistochemical evaluation of noncol-

lagenous proteins of bone (osteonectin, osteopon-

tin, and osteocalcin) may in some cases help to

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

Supported by: European Commission, Grant number:EuroBoNeT LSHC-CT-2006-018814; Jose Maria Buesa 2007 fromthe Spanish Group for Research on Sarcomas.

*Correspondence to: Tarja Niini, Laboratory of CytomolecularGenetics, Department of Pathology, HUSLAB, P.O. Box 400,FI-00029 HUS, Helsinki, Finland. E-mail: tarja.niini@helsinki.fi

Received 28 May 2009; Accepted 23 September 2009

DOI 10.1002/gcc.20727

Published online 27 October 2009 inWiley InterScience (www.interscience.wiley.com).

VVC 2009 Wiley-Liss, Inc.

distinguish these three tumor types (Serra et al.,

1996).

Very little is understood regarding the genetics

of FS of bone. To our knowledge, only two

papers have previously been published about this

topic. We previously studied nine FSs of bone by

conventional comparative genomic hybridization

(CGH) and array CGH using microchips contain-

ing probes specific for 59 oncogenes and tumor

suppressor genes (Hattinger et al., 2004). Hallor

et al. (2007) studied one case of FS of bone using

cytogenetics, fluorescence in-situ hybridization

(FISH), and array CGH encompassing about

30,000 BAC clones.

In this study, we performed an array CGH

analysis to find genomic regions or genes with

potential involvement in the pathogenesis of FS

of bone. Nine primary tumors and four local

recurrences from 11 patients were studied using

CGH oligo microarrays containing 244,000 probes

encompassing the whole genome.

MATERIALS AND METHODS

Patients and Material

Nineteen fresh frozen tissue specimens from

17 patients with FS of bone were considered for

this study (15 primary tumors and 4 local recur-

rences). After histological revision, tumor speci-

mens with only a proportion of neoplastic cells

over 80% were used for DNA isolation. Enough

good quality DNA for array CGH hybridization

was available from 13 of these specimens (nine

primary tumors and four local recurrences)

obtained from 11 patients. The supplementary

analyses for methylation and mutations were per-

formed on 16 specimens (12 primary tumors and

4 local recurrences) from 14 patients, as quality

and amount of DNA in these analyses is not as

critical as in array CGH. All clinical data of the

whole group of 17 patients are shown in Table 1.

DNA Isolation

Genomic DNA was extracted using the stand-

ard phenol–chloroform method. Two reference

DNAs, female and male, were extracted from

pools of blood samples (four individuals per

each). The concentration and the quality of DNA

were measured using NanoDrop ND-1000 Spec-

trophotometer (NanoDrop Technologies, Wil-

mington, DE) and gel electrophoresis.

Array CGH Hybridization and Data Analysis

Digestion, labeling, and hybridization were

performed by following Agilent’s protocol version

4.0 for Agilent Human Genome CGH 244A oligo

microarrays (Agilent Technologies, Santa Clara,

CA). Briefly, same amounts (1.5 lg) of patient

DNA and gender-matched reference DNA were

digested. The digested DNAs were labeled by

random priming with Cy3-dUTP (reference

DNA) and Cy5-dUTP (patient DNA) using Agi-

lent Labeling Kit. The labeled DNAs were puri-

fied. Afterward, differentially labeled patient and

reference DNAs were combined and hybridized

to Agilent Human Genome CGH 244A microar-

rays at 65�C for 40 hr. The hybridized arrays

were washed and scanned using Agilent scanner.

The array images were analyzed using Agilent

Feature Extraction Software (version 9.5.3.1),

which also performs dye normalization for the

data. The normalized data were analyzed using

Agilent DNA Analytics Software (version 4.0)

with ADM-2 algorithm, which is an aberration

detection algorithm that identifies all aberrant

intervals in a given sample with consistently high

or low log ratios based on the statistical score. In

addition, it incorporates quality information about

each probe measurement. The ADM-2 algorithm

searches for intervals in which the average log ra-

tio of the sample and reference channels exceed

a user specified threshold. In this study, we used

threshold ‘‘8.’’ To get rid of long aberrations with

low absolute mean ratios that often represent

noise, we applied Fuzzy Zero correction algo-

rithm. Fuzzy Zero applies a correction to all aber-

rant intervals identified in ADM-2 analysis by

reassessing their significance without the assump-

tion of probe-to-probe error independence.

The minimum log ratio that the ADM-2 algo-

rithm considers as a gain or a loss depends on the

quality of the hybridization and the number of

the probes in the region. To assure the exclusion

of very low or very small aberrations, we added a

custom-made aberration filter defining a region

with a copy number aberration as a region with

minimum seven probes gained/lost and with min-

imum absolute average log2 ratio of 0.2 (gain) or

�0.2 (loss). The level of losses and gains was

checked visually in the individual profiles. A loss

was regarded as homozygous when log2 ratio was

lower than �1 and a gain as high level amplifica-

tion when log2 ratio was higher than 1.

The smallest somatic copy number variations

were automatically excluded because of the

COPY NUMBER PROFILING IN FIBROSARCOMA OF BONE 133

Genes, Chromosomes & Cancer DOI 10.1002/gcc

TABLE1.ClinicopathologicFeaturesof17Patients

withFibrosarcomaofBone

Patient

sample

analyzed

atdiagnosis

(years)

Gender

Histologic

primary

Treatment

Typeofsurgery

Surgical

margins

months

untilfirst

relapse

Typeof

firstrelapse

months

untillast

follow

outcome

1priþ

Female

4Femur

Surgery

Resection

Localrecurrence

4Tibia

Surgery

Resection

Localrecurrence

4priþ

4Tibia

Surgery

Amputation

Radical

Metastasisin

softtissue

Female

4Humerus

Surgery

Resection

7Lungmetastasis

4Femur

Surgery

þpost-opCH

Amputation

9Localrecurrence

þlungmetastasis

3Pelvis

Surgery

Resection

3Pelvis

Surgery

Resection

Intralesional

7Localrecurrence

3Scapula

Pre-opCH

þsurgery

þpost-opCH

Resection

4Humerus

Surgery

þpost-opCH

Resection

Lungmetastasis

atonset

4Pelvis

Pre-opCH

þradiotherapy

þsurgery

Amputation

Marginal

7Lungmetastasis

3Femur

Surgery

Resection

2Lungþ

metastasis

4Pelvis

Pre-opCH

þsurgery

þpost-opCH

Resection

Secondtumor

4Pelvis

Chemotherapy

Notdone(refused

bythepatient)

Female

4Humerus

Surgery

Amputation

Lungmetastasis

atonset

Female

3Pelvis

Surgery

Resection

Marginal

atfollow

Female

4Femur

Surgery

þpost-opCH

Resection

Lungmetastasis

3Fibula

Pre-opCH

þsurgery

þpost-opCH

Resection

pri,primarytumor;

localrecurrence;NED,noevidence

ofdisease;NED2,noevidence

ofdiseaseafterrelapse;AW

D,alivewithdisease;DOD,dead

ofdisease;pre-opCH,preoperative

chemotherapy;

post-opCH,postoperative

chemotherapy.

134 NIINI ET AL.

custom-made aberration filter (minimum seven

probes gained/lost). For the regions with most

frequent gains or losses, the somatic copy number

variations were excluded by visual examination of

the regions, with a connected window of known

copy number variable regions in the DNA Ana-

lytics Software.

Methylation-Specific Polymerase Chain Reaction of

the p16INK4a and p14ARF Genes

Methylation-specific PCR for p16INK4a and

p14ARF promoter methylation was performed as

described previously (Herman et al., 1996; Estel-

ler et al., 2000). The DNA was modified with

bisulphite solution by using ImprintTM DNA

Modification Kit (Sigma-Aldrich, St Louis, MO)

according to the manufacturer’s instructions. After

bisulphite modification, specific primers recogniz-

ing the methylated and unmethylated sequences

of the promoter regions of p16INK4a and p14ARF

were used for methylation-specific PCR as previ-

ously reported (Herman et al., 1996; Esteller

et al., 2000).

The PCR mixture contained �30 ng of bisul-

phite-treated DNA, 0.2 mM deoxynucleotide tri-

phosphates, 1.5 mM magnesium chloride, 20 pmol

of each primer, 1� PCR buffer P, and 1 unit of

AmpliTaq Gold (Applied Biosystems, NJ, USA)

in a final volume of 20 ll. Amplification was car-

ried out in a PTC-200 Peltier Thermal Cycler

(BIO-RAD, CA, USA). Reactions of p16INK4a

were denatured for 15 min at 95�C and incubated

for 35 cycles [95�C for 30 s, 60�C (unmethylated)

or 65�C (methylated) for 30 s, 72�C for 30 s] and

followed by a final 10 min extension at 72�C.Reactions of both unmethylated and methylated

PCR for p14ARF were denatured for 15 min at

95�C and incubated for 35 cycles (95�C for 45 s,

58�C for 45 s, 72�C for 1 min) and followed by a

final 10 min extension at 72�C. Each PCR prod-

uct was loaded on a 1.5% agarose gel, stained

with ethidium bromide, and visualized under UV

illumination.

Mutational Analysis of KIT, PDGFRA, and KDR

Intronic PCR primers were used to amplify

Exons 9, 11, 13 (Lasota et al., 2000), and 17 of

KIT (Corless et al., 2002) and Exons 12 and 18 of

PDGFRA (Heinrich et al., 2003). Primers for

exons codifying the yuxtamembrane and kinase

domains of KDR were designed (Supporting In-

formation Table 1). PCR was performed in 25 ll

reactions containing 2 ll DNA, 50 mM KCl, 10

mM Tris–HCl, pH 8.3, 2 mM MgCl2, 0.2 mM of

each dNTP, 0.2 lM for each primer, and 2 U

AmpliTaq Gold (Perkin-Elmer, Norwalk, CT).

PCR was carried out in a DNA Thermal Cycler

9700 (Perkin-Elmer, Norwalk, CT) wherein the

samples were preheated in an initial step at 95�Cfor 10 min; amplified by 40 cycles of 1 min of

denaturation at 94�C, 1.5 min of annealing at

56�C for KIT primers, 2 min at 65�C for PDGFRAprimers or 1 min at 58�C for KDR primers, and 1

min of extension at 72�C. An additional extension

step of 10 min was also performed. Ten-microli-

ter PCR products were visualized on an ethi-

dium-bromide-stained 2% ultraPure agarose gel

electrophoresis (Life Technologies, Paisley, Scot-

land) and photographed. Negative controls were

included in every set of amplifications. A bidirec-

tional sequencing analysis on an ABI 3130xl

sequencer using the BigDye terminator v3.1 kit

(Applied Biosystems, Foster City, CA) with the

specific primers was performed. Ensembl (http://

www.ensembl.org/Homo_sapiens/index.html) con-

sensus sequences for KIT (ENSG00000157404),

PDGFRA (ENSG00000134853), and KDR (ENS-

G00000128052) were used for characterizing the

mutations.

Immunohistochemistry of KIT, PDGFRA,

and PDGFRB

The expression of KIT, PDGFRA, and

PDGFRB was studied by immunohistochemistry

in the 19 tumor specimens (15 primary tumors

and 4 local recurrences) obtained from the whole

group of 17 patients originally considered for this

study. By using an avidin–biotin-peroxidase com-

plex method, immunohistochemistry was carried

out with the following primary antibodies: antihu-

man CD117 (KIT) rabbit polyclonal antibody

(1:50 dilution rate; DakoCytomation, Glostrup,

Denmark), antihuman PDGF receptor a-subunit(1:100 dilution rate), and antihuman PDGF re-

ceptor b-subunit (1:200 dilution rate) mouse

monoclonal antibodies (both from Genzyme,

Cambridge, MA). Primary antibodies were incu-

bated overnight at 4�C. Development of immu-

noreaction was obtained with diaminobenzidine

and nuclei were counterstained with Gill’s hema-

toxylin. As positive controls, tissue sections of a

PDGF receptor-positive breast cancer and of a

KIT-positive gastrointestinal tumor were used.

Immunostaining was scored on a scale of positiv-

ity from one plus to three plus, according to the

percentage of positive cells. Cases with 5–10%

positive cells were classified as þ, cases with 10–

50% positive cells as þþ, and cases with more

than 50% positive cells as þþþ. All immunohis-

tochemical stainings were evaluated without

knowing the genetic data to avoid any possible

bias in data interpretation.

RESULTS

Array CGH Findings

The original data of the array CGH hybridiza-

tions of all the samples can be found in public

database Cangem (http://www.cangem.org/). The

overview of the copy number changes of all

patients along the whole genome is shown in Fig-

ure 1. In Figure 1, as well as in the text below,

from the patients with two specimens (Patients 1

and 4), only the primary tumor is included in the

overall frequencies for the 11 patients. Losses

and gains were approximately equally common.

The numbers of aberrations per sample are

shown in Table 2 and ranged from 0 to 132, with

an average of 43 for the eleven patients.

The minimal overlapping regions of most fre-

quent losses are shown in Table 3. The regions

at 9p21.3, 20p12.3, and 20p12.2-p11.23 were lost

in 7 (64%) and the regions at 6q13-q23.2, 8p23.3,

8p23.2, 13q21.31-q32.2, and 20p13 in 6 (55%) of

the 11 patients.

Closer examination of the region at 9p21.3

(21,973,069–22,007,894) revealed that seven

patients (64%) had a homozygous deletion with

minimal overlapping region at the CDKN2A[cyclin-dependent kinase inhibitor 2A (mela-

noma, p16, inhibits CDK4)] locus (Table 2). In

three patients, the homozygous deletion encom-

passed only part of CDKN2A, in two patients

affecting the 30 end of the gene (21,733,410–

21,980,581 in Patient 7; 21,919,810-21,973,128 in

Patient 13), and in one patient the 50 end of the

gene (21,958,041–22,007,894 in Patient 3). The

CDKN2B [cyclin-dependent kinase inhibitor 2B

(p15, inhibits CDK4)] locus was included in six

(Table 2) and the MTAP (methylthioadenosine

Figure 1. The penetrance overview of array CGH findings in 11 patients with fibrosarcoma of bone.Copy number losses are shown by green color on the left and copy number gains by red color on theright of the vertical line. Note: Losses and gains consequent upon copy number variants are included inthe figure. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

136 NIINI ET AL.

TABLE2.To

talNumberofDNACopy

NumberChanges,Copy

NumberofaSelectedGroupofTumorSuppressorGenesandOncogenes,andIm

munohistochemistryResultsofKIT,

A,andPDGFR

Patients

withFibrosarcomaofBone

Patient

findings

Immunohistochemistryfindings

changes

CDKN2A

CDKN2B

STARD13

KIT,PD

PDGFRB

APDGFR

þþ�

hom.loss

þþþ

�Neg

hom.loss

þ��

hom.loss

þ��

hom.loss

Nhet.loss

þþ�

�Neg

þ��

hom.loss

het.loss

þþ�

hom.loss

Nhom.loss

þþ�

þþþ

þ��

hom.loss

Nhet.loss

het.loss

pri,primarytumor;rec,

localrecurrence;N,norm

al;NA,notavailable;Neg,negative;hom.loss,homozygousloss

(log 2

ratio�

�1);het.loss,heterozygousloss;am

p,highlevelam

plification(log 2

ratio�

þ��,

weaklypositive

(5–10%

positive

tumorcells);þþ

�,moderatelypositive

(10–50%

positive

tumorcells);þþ

þ,highlypositive

positive

tumorcells).

Entriesin

thefirsttw

orows(Patientcode1,priandrec)

andthefourthandfifthrows(Patientcode4,priandrec)

identify

tumorlesionsfrom

thesamepatient.

Localrecurrencesarelistedin

phosphorylase) locus in four of the homozygous

deletions (Patients 4, 7, 11, and 16).

Another small region frequently lost (in 5/11

patients; 45%) was found at 13q13.1 (32,690,731–

32,734,316). Closer examination revealed that the

minimal common region came from an internal

homozygous deletion of the tumor suppressor

gene STARD13 [StAR-related lipid transfer

(START) domain containing 13; DLC2] in one of

the patients (Patient 16; Table 2). The four het-

erozygous losses containing the gene encom-

passed the whole or most of the chromosome.

The minimal overlapping regions of most fre-

quent gains are shown in Table 3. The regions at

4q12, 12p13.1-p12.1, and Xp11.4-p11.1 were

gained in 7 (64%), and the regions at 1q21.1,

4q13.1, 5p14.3-p12, 8q12.3-q21.11, 22q11.21-

q11.22, 22q12.1-q13.1, and 22q13.31 in 6 (55%) of

the 11 patients. Altogether, ten high-level ampli-

fications were detected in 4 of the 11 patients

(36%), located at the regions 4p14 (36,988,420–

39,352,822) (Patient 4, local recurrence), 4q12

(52,383,858–59,121,895 in Patient 11; 52,383,858–

59,422,728 in Patient 13), 7p22.3-p21.3 (140,

213–10,140,104), 10q22.3 (75,812,637–79,624,210)

(Patient 4, primary tumor), Xq22.1 (98,731,361–

101,121,952), Xq27.3 (142,333,851–142,953,478

and 146,486,322–146,822,718), and Xq28

(149,920, 971–151,352,118 and 151,962,182–

152,799,627) (Patient 13). The region at 4q12,

harboring KIT (v-kit Hardy–Zuckerman 4 feline

sarcoma viral oncogene homolog), PDGFRA (pla-

telet-derived growth factor receptor, alpha poly-

peptide), and KDR [kinase insert domain receptor

TABLE 3. The Minimal Overlapping Regions of Most Common Copy Number Losses and Most Common Copy Number GainsDetected by Array CGH in 11 Patients with Fibrosarcoma of Bone

Chromosomal location Start (bp) Stop (bp) Size No. of patients

Most Common Copy Number Losses6q13-q23.2 75,407,630 131,484,077 56.1 Mb 68p23.3 ,166,452 1,793,590 1.6 Mb 68p23.2 3,470,621 4,892,845 1.4 Mb 68p21.3-p11.21 21,198,243 43,315,378 22.1 Mb 59p24.3-p23 ,194,193 12,239,566 12.0 Mb 59p22.2 18,176,747 18,613,764 437.kb 59p21.3 21,973,069 22,007,894 35.kb 79p21.2 26,812,713 26,918,415 106.kb 59p21.2-p21.1 27,374,534 29,245,382 1.9 Mb 510p11.22-p11.21 34,272,515 35,513,346 1.2 Mb 510q21.1-q21.3 53,139,879 68,349,211 15.2 Mb 513q12.12-q12.3 24,239,490 28,397,462 4.2 Mb 513q13.1 32,327,843 32,504,385 177.kb 513q13.1 32,690,731 32,734,316 44.kb 513q21.1-q21.2 52,065,165 57,939,368 5.9 Mb 513q21.31-q32.2 60,904,588 97,598,765 36.7 Mb 620p13 ,369,374 2,462,672 2.1 Mb 620p13 4,107,774 4,663,610 556.kb 620p12.3 6,747,437 8,268,916 1.5 Mb 720p12.2-p11.23 11,731,438 20,217,333 8.5 Mb 7

Most common copy number gains1q21.1 145,009,467 147,499,046 1.3 Mb 64q12 52,424,522 59,485,323 7.1 Mb 7a

4q13.1 60,726,221 62,670,198 1.9 Mb 65p14.3-p12 19,422,745 43,774,710 24.4 Mb 68q12.3-q21.11 65,458,034 76,508,544 11.1 Mb 612p13.1-p12.1 13,851,257 34,108,730 20.3 Mb 715q26.3 96,907,698 97,551,434 644.kb 516q22.1 65,432,627 67,587,824 2.2 Mb 517q23.3-q24.2 58,392,690 64,014,945 5.6 Mb 520q11.21-q11.22 29,436,537 31,850,249 2.4 Mb 520q11.23-q13.33 35,260,840 58,435,010 23.2 Mb 522q11.21-q11.22 16,299,747 21,544,846 5.2 Mb 622q12.1-q13.1 27,128,362 37,962,936 10.8 Mb 622q13.31 45,056,333 45,616,686 560.kb 6Xp11.4-p11.1 42,159,896 58,068,490 15.9 Mb 7

aIncluding two patients with a high level amplification.

138 NIINI ET AL.

(a type III receptor tyrosine kinase); VEGFR2],was highly amplified in 2 of the 11 patients (18%;

Table 2).

A small minimal overlapping region of gains at

15q26.3 (96,907,698–97,551,434) contains four

genes, one of which is IGF1R (insulin-like growth

factor 1 receptor). This region was gained in 5 of

the 11 patients (45%; Table 2). Another relatively

small minimal overlapping region at 1q21.1

(145,009,467–147,499,046), gained in 6 of the

patients (55%), contains 12 genes. One of these is

CHD1L (chromodomain helicase DNA-binding

protein 1-like; Table 2).

There were four regions showing clearly higher

frequency of gains in the local recurrences (three

of four; 75%) compared to the overall frequency.

The minimal overlapping regions of these gains

were seen at 7q35-q36.1, 8q23.3-q24.13, 11q12.2-

q12.3, and 11q13.2-q13.3 (Patients 3, 4, and 7).

Because of the small number of patients, the sta-

tistical significance of difference in frequencies

could not be demonstrated. The region at

11q13.2-q13.3 (66,875,572–70,410,288; size 3,5

Mb) contains the CCND1 (cyclin D1) gene and

FGF (fibroblast growth factor) cluster with around

50 other genes.

Hypermethylation of CDKN2A

Because of the finding of frequent deletion of

CDKN2A in array CGH, methylation status of the

two genes at the CDKN2A locus, p16INK4a and

p14ARF, was studied in 16 specimens from 14

patients (12 primary tumors and 4 local recur-

rences). Since array CGH fundamentally is not a

quantitative method, the methylation analysis

was also performed on the seven patients (eight

samples), which seemed to possess a homozygous

deletion at CDKN2A. The other seven patients

included four patients (five samples) with no de-

letion at CDKN2A and three patients without

array CGH result. Hypermethylation of both

p16INK4a and p14ARF was detected in 1 of the 14

patients studied (7%; Patient 9), a patient lacking

an array CGH result. Both genes were hyperme-

thylated in this same patient.

Mutational Analysis of KIT, PDGFRA, and KDR

The KIT, PDGFRA, and KDR genes, located in

the region with recurrent high level amplification,

were screened for mutations in 16 tumor speci-

mens (12 primary tumors and 4 local recurrences)

from 14 patients. Particular regions that codify for

the yuxtamembrane and kinase domains, crucial

for the regulation of the activity of these tyrosine

kinase receptors, were studied. No activating

mutations were found in these regions in any of

the genes. One patient (Patient 15) without array

CGH result showed a heterozygous silent muta-

tion in the third position of Codon 798 of KIT[ATC (Ile) > ATT (Ile)] (Exon 17), which is not

a polymorphism. Four patients (Patients 4, 7, 10,

and 15) carried the polymorphism rs2228230 in

Exon 18 of PDGFRA. This polymorphism was

present heterozygously and consisted of a synony-

mous change in the third position of Codon 824

[GTC (Val) > GTT (Val)].

Immunohistochemistry of KIT, PDGFRA,

and PDGFRB

KIT and PDGF-receptors were selected to be

studied by immunohistochemistry, since the KITand PDGFRA genes were included in the region

with recurrent high level amplification. The

results are shown in Table 2.

KIT was expressed in 6 of the 15 primary

tumors (40%) and in one of the four local recur-

rences (25%). Among the seven cases that

showed a KIT-positive immunoreaction, the 4q12

region was highly amplified in two patients

(Patients 11 and 13), gained in two patients

(Patients 4 and 16), and not affected by changes

in two patients (Patients 1 and 12). In one of the

patients, array CGH result was missing.

PDGFRA was expressed in 6 of the 13 pri-

mary tumors with available immunohistochemis-

try data (46%) and in all of the four local

recurrences. Among the seven tumors showing

gain of 4q12 region, a positive immunoreaction

for PDGFRA was present in four cases (Patients

3, 4, 6, and 16). PDGFRB was also expressed in

6 of the 13 primary tumors (46%) and in all local

recurrences. Immunohistochemistry data posi-

tively correlated with array CGH results: both

two cases with losses at 5q (Patients 13 and 18)

were negative for PDGFRB and both two cases

with gain at 5q were PDGFRB-positive (Patient

4, recurrence and Patient 16). However, there

were seven cases in which PDGFRB positivity

was not associated with changes at 5q. By consid-

ering the 12 cases that resulted positive to either

PDGFRA or PDGFRB, it is worthwhile noting

that eight cases (including all four local recur-

rences) expressed both receptors and four cases

only one of them.

DISCUSSION

High number of recurrent copy number aberra-

tions was detected in the 11 patients with FS of

bone. Gains were most frequently found at

regions 1q, 4q, 5p, 8q, 12p, 15q, 16q, 17q, 20q,

22q, and Xp, each of which has previously been

found to be commonly gained in at least some

types of sarcomas (Mairal et al., 1999; Pandita

et al., 1999; Squire et al., 2003; Lau et al., 2004;

Man et al., 2004; Larramendy et al., 2006; Meza-

Zepeda et al., 2006; Ohguri et al., 2006; Rozeman

et al., 2006; Tarkkanen et al., 2006). The regions

with most common losses were 6q, 8p, 9p, 10,

13q, and 20p. All these regions have been found

to be frequently lost in many types of sarcomas,

except for 20p, which seems to be relatively com-

mon only in MFHs of soft tissue and bone (Mai-

ral et al., 1999; Tarkkanen et al., 2006).

In our previous study (Hattinger et al., 2004),

which included a different series of FS of bone

patients, the most frequently detected aberrations

were gains of the chromosomal region 22q (by

conventional CGH) and of the platelet-derived

growth factor beta (PDGFB) gene located at

22q12.3-q13.1 (by array CGH). Findings obtained

in the present study were in agreement with

those already published and further indicated the

involvement of 22q harbored genes in the patho-

genesis of FS of bone. In the other previous

study of FS of bone, Hallor et al. (2007) studied

only one case with ring chromosome 6 and found

losses of distal ends of 6p and 6q as sole copy

number aberrations using array CGH.

When comparing our results to the pattern of

changes found in array CGH studies of OS

(Squire et al., 2003; Lau et al., 2004; Man et al.,

2004; Zielenska et al., 2004; Atiye et al., 2005),

many similarities were seen, but also many differ-

ences could be pointed out, the most striking

ones at the regions 3p, 6p, 8p, 12p, 17p, 20p, and

22. More than the changes in OS, our results

resembled the pattern of changes detected in 26

patients with MFH of bone using conventional

CGH (Tarkkanen et al., 2006). Differences, how-

ever, could also be found compared to results of

that study, the most eye-catching ones at the

regions 1p, 6p, and 12q with more frequent gains

in the patients with MFH and the region 8p with

more frequent losses in our patients with FS.

The range in the number of aberrations per

patients was high. In three patients, none or only

a couple of aberrations were seen. In the other

eight patients, the number of alterations ranged

from 20 to 132, with an average of 58. Thus, as

in many other sarcoma types (Mairal et al., 1999;

Pandita et al., 1999; Squire et al., 2003; Larra-

mendy et al., 2006; Rozeman et al., 2006; Tarkka-

nen et al., 2006), high chromosomal instability is

a common phenomenon in FS of bone, too. The

possible association between complexity of copy

number karyotype and prognosis needs to be

studied with a larger patient material.

Homozygous losses with minimal overlapping

region at the CDKN2A locus (9p21.3) were

detected in 7 of the 11 patients (64%). Six of the

losses also included CDKN2B. Our results suggest

that in FS of bone the homozygous deletions of

CDKN2A are much more common than in OS, in

which they are seen in about 10% of the patients

(Miller et al., 1996; Nielsen et al., 1998; Wei

et al., 1999; Tsuchiya et al., 2000; Patino-Garcia

et al., 2003; Lopez-Guerrero et al., 2004). Homo-

zygous deletions of the gene have also been

reported in MFH of soft tissue (Orlow et al.,

1999; Simons et al., 2000). To our knowledge, al-

together nine cases with FS of soft tissue have

been studied for the deletions at CDKN2A, but

no deletions were found (Maelandsmo et al.,

1995; Orlow et al., 1999).

CDKN2A encodes two different transcripts,

p16INK4a and p14ARF, from different promoters

and in alternate reading frames [for review, see

Kim and Sharpless (2006)]. Methylation status of

p16INK4a and p14ARF was studied in 14 patients,

including 7 patients showing a homozygous dele-

tion of CDKN2A in array CGH. Both p16INK4a

and p14ARF were found to be hypermethylated in

one and the same patient (a patient without an

array CGH result). Deletion, thus, seems to be

the main mechanism of the inactivation of these

genes in FS of bone. Though hypermethylation

of both p16INK4a and p14ARF was detected in the

same patient, methylation of these two genes has

been reported to occur independently in colo-

rectal carcinoma (Esteller et al., 2000).

It is noteworthy that all the patients with the

loss of CDKN2A possessed high number of copy

number changes (29–132). This suggests that the

mechanisms driving tumorigenesis in patients

with a close to normal karyotype may be totally

different from those in the patients with a com-

plex karyotype.

Interestingly, the CCND1 gene encoding cyclin

D1 that acts in the same pathway as p16INK4a

with opposing effects, is situated at region

11q13.2-q13.3 that was gained in three of the

four local recurrences (75%). Concerning the RB1

140 NIINI ET AL.

gene, heterozygous large losses at 13q, mostly

affecting the whole chromosome, were found in 4

of the 11 patients (36%). All of the patients with

gain at CCND1 or loss at RB1 also showed a

homozygous deletion at CDKN2A. Regarding

TP53, a 72-kb homozygous deletion containing

the gene (17p13.1; 7,466,188–7,537,896) was

found in one of the patients (Table 2). Thus, it

seems that not only the disruption of RB pathway

but also that of TP53 pathway plays a role in the

pathogenesis of FS of bone.

Single recurrent high level amplification

located at 4q12 was found in our patient material.

This region has previously been shown to be fre-

quently gained in OS (Squire et al., 2003; Atiye

et al., 2005) and in MFH of bone (Tarkkanen

et al., 2006). In OS, high level amplifications at

4q12 have also been reported (Lau et al., 2004;

Man et al., 2004). The region harbors the genes

encoding tyrosine kinases KIT, PDGFRA, and

KDR, which are of particular interest because of

their potential use as targets for directed thera-

pies. These genes were screened for mutations in

14 of our patients, but no activating mutations

were found in any of the genes.

The absence of KIT mutations was expectable

as, to our knowledge, KIT has not been reported

to be activated by mutations in other sarcomas

than GIST [Burger et al., 2005; reviewed in

Miettinen and Lasota (2005)]. Expression of KIT

was, however, detected in 5 of our 15 primary

tumors (33%) and in one of four local recurrences

(25%). The samples with high level amplification

at 4q12 showed moderate expression of the pro-

tein. Overexpression of the wild-type KIT recep-

tors has been reported in many neoplasms,

including OS and Ewing sarcoma (Tamborini

et al., 2004; Burger et al., 2005; Entz-Werle et al.,

2005; McIntyre et al., 2005; Bozzi et al., 2007).

Fibroblastic soft tissue sarcomas, as most soft tis-

sue sarcomas, have generally been found to be

KIT negative [Hornick and Fletcher, 2002;

reviewed in Miettinen and Lasota (2005)]. In a

study of OS, KIT overexpression resulted from

amplification of the gene itself in most of the

cases (Entz-Werle et al., 2007). In other tumors,

including Ewing sarcoma, the overexpression of

the wild-type receptor has usually been suggested

to be caused by autocrine/paracrine loop activa-

tion (Tamborini et al., 2004; Burger et al., 2005;

Bozzi et al., 2007). In many cases, the phospho-

rylation of KIT has also been studied, and in

each example the wild-type KIT receptor was

shown to be phosphorylated and, thus, as its

active form (Gonzalez et al., 2004; Tamborini

et al., 2004; Bozzi et al., 2007). Based on these

data, we can hypothesize that our samples are

also expressing a phosphorylated and active KIT

receptor.

PDGFRA was expressed in 6 (46%) and

PDGFRB also in 6 (46%) of the 13 primary

tumors. Coexpression of the receptors was

detected in 4 of the 13 primary tumors (31%) and

in all of the local recurrences. The expression of

PDGFRA and PDGFRB in more than half of the

samples, as well as their frequent coexpression,

including all of the local recurrences, supports

our previous study that already suggested these

receptors to play an important role in the patho-

genesis of FS of bone (Hattinger et al., 2004).

We previously detected expression of PDGFRA

in 20%, the expression of PDGFRB in 28%, and

simultaneous expression of PDGFB ligand and

the receptors in 32% of 25 cases with FS of bone,

suggesting a presence of a PDGFB-mediated

autocrine loop. Using conventional CGH, we also

found a gain at the chromosomal region 22q con-

taining the PDGFB gene (22q13.1) in seven of

nine cases (78%), including one high level ampli-

fication. In addition, we showed a gain of PDGFBin three of five cases using DNA arrays. These

results are in concordance with our present study

showing a gain at the region containing PDGFBin 6 of the 11 patients (55%).

The 4q12 region with high level amplifications

in our patients also contains the KDR gene.

Overexpression of its ligand, VEGF, associates

with poor prognosis in sarcomas, including OS

and Ewing sarcoma [Lee et al., 1999; Kaya et al.,

2000; Fuchs et al., 2004; reviewed in DuBois

and Demetri (2007)]. In one study of 30 patients

with nonmetastatic OS, the expression of KDR

was found in 67% of the patients but the expres-

sion did not correlate with survival (Lee et al.,

1999).

Frequent losses (in 5/11 patients; 45%) were

found at 13q13.1 with minimal overlapping region

inside the STARD13 gene. The small region

derived from a homozygous internal deletion of

the gene in one of the patients. STARD13 was

recently identified as a tumor suppressor gene in

hepatocellular carcinoma (Leung et al., 2005),

and it has also been found to be downregulated

in many other solid tumors (Ullmannova and

Popescu, 2006). The gene encodes a GTPase-

activating protein exhibiting its tumor suppressor

functions by negatively regulating RHOA (ras

homolog gene family, member A), the

overexpression of which is believed to be

involved in tumorigenesis of many cancer types

[Pille et al., 2005; reviewed in Sahai and Marshall

(2002)].

A small minimal overlapping region of frequent

gains (in 5/11 patients; 45%) was detected at

15q26.3. The region contains only four genes,

one of which is IGF1R. The IGF1R pathway has

been shown to play a central role in sarcomas,

and the first clinical trials targeting IGF1 receptor

in sarcoma therapy are currently underway

[reviewed in Scotlandi and Picci (2008)]. The

15q26.3 region with IGF1R is frequently gained

in chondrosarcoma and MFHs of bone and soft

tissue (Mairal et al., 1999; Rozeman et al., 2006;

Tarkkanen et al., 2006). Moreover, one high-level

amplification of the 15q25-qter region has been

reported in OS (Squire et al., 2003). If IGF1Rturns out to be important for the pathogenesis of

FS of bone, as our results suggest, the patients

can be assumed to benefit from treatment tar-

geted to IGF1R in this sarcoma type, too.

Another relatively small minimal overlapping

region of frequent gains (in 6/11 patients; 55%)

was found at the region 1q21.1. This region is

frequently gained in many solid tumors, including

OS and MFHs of both bone and soft tissue

(Simon et al., 1998; Tirkkonen et al., 1998; Fang

et al., 2001; Squire et al., 2003; Lau et al., 2004;

Atiye et al., 2005; Tarkkanen et al., 2006; Ma

et al., 2008). Moreover, high level amplifications

have been reported in a few patients with OS

(Lau et al., 2004; Atiye et al., 2005) and one

patient with MFH of soft tissue (Mairal et al.,

1999). The minimal overlapping region of the

gains in our patients contains 12 genes. Interest-

ingly, one of these is CHD1L, which was recently

reported as a candidate oncogene in hepatocellu-

lar carcinoma (Ma et al., 2008).

In conclusion, our array analysis revealed

several regions and genes, which may be involved

in the pathogenesis of FS of bone. Particularly,

the deletion of CDKN2A seems to play a crucial

role in a substantial proportion of the patients.

The recurrent high level amplification at 4q12

including KIT, PDGFRA, and KDR suggests that

some, if not all, of these genes may also have

influence on the pathogenesis of this tumor.

Other genes with potential importance include

STARD13, IGF1R, and CHD1L. The results open

up a good starting point for identifying new

diagnostic and prognostic markers, as well as

potential targets for therapy in this aggressive

type of tumor.

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Frequent deletion of CDKN2A and recurrent coamplification of KIT, PDGFRA, and KDR in fibrosarcoma of...

Documents

Microfluidic Deletion/Insertion Analysis for Rapid Screening of KIT and PDGFRA Mutations in CD117-Positive Gastrointestinal Stromal Tumors

Use of c-KIT/PDGFRA mutational analysis to predict the clinical response to imatinib in patients with advanced gastrointestinal stromal tumours entered on phase I and II studies of

Glucose is a key regulator of VEGFR2/KDR in human epithelial ovarian carcinoma cells

p16 Inactivation by methylation of the CDKN2A promoter occurs early during neoplastic progression in Barrett's esophagus

Sodium channel genes and their differential genotypes at the L-to-F kdr locus in the mosquito Culex quinquefasciatus

Functional evaluation of tumour-specific variants of p16INK4a/CDKN2A: correlation with protein structure information

Kaposi's sarcoma associated herpesvirus G protein-coupled receptor immortalizes human endothelial cells by activation of the VEGF receptor-2/ KDR

Cooperative interactions of BRAFV600E kinase and CDKN2A locus deficiency in pediatric malignant astrocytoma as a basis for rational therapy

FIP1L1-PDGFRA molecular analysis in the differential diagnosis of eosinophilia

A Flexible Multiplex Bead-Based Assay for Detecting Germline CDKN2A and CDK4 Variants in Melanoma-Prone Kindreds

Identification of a Variant in KDR Associated with Serum VEGFR2 and Pharmacodynamics of Pazopanib

Prevalence and predictors of germline CDKN2A mutations for melanoma cases from Australia, Spain and the United Kingdom

Oncogenic and ligand-dependent activation of KIT/PDGFRA in surgical samples of imatinib-treated gastrointestinal stromal tumours (GISTs)

Loss of heterozygosity of CDKN2A (p16INK4a) and RB1 tumor suppressor genes in testicular germ cell tumors

Predicting functional significance of cancer-associated p16INK4a mutations in CDKN2A

CDKN2A/B Alterations Impair Prognosis in Adult BCR-ABL1-Positive Acute Lymphoblastic Leukemia Patients

Intestinal Neurofibromatosis Is a Subtype of Familial GIST and Results From a Dominant Activating Mutation in PDGFRA

EXPRESSION OF VEGF AND ITS RECEPTORS FLT1 AND FLK-1/KDR ARE ALTERED IN LAMBS WITH INCREASED PULMONARY BLOOD FLOW AND PULMONARY HYPERTENSION

Occurrence of the leucine-to-phenylalanine knockdown resistance (kdr) mutation in Anopheles arabiensis populations in Tanzania, detected by a simplified high-throughput SSOP-ELISA

A Tyrosine Kinase Created by Fusion of the PDGFRA and FIP1L1 Genes as a Therapeutic Target of Imatinib in Idiopathic Hypereosinophilic Syndrome