Array-CGH identiﬁes cyclin D1 and UBCH10 amplicons in ...erc.endocrinology-journals.org/content/15/3/801.full.pdf · Array-CGH identiﬁes cyclin D1 and UBCH10 amplicons in anaplastic

Endocrine-Related Cancer (2008) 15 801–815

Array-CGH identifies cyclin D1 and UBCH10amplicons in anaplastic thyroid carcinoma

Jia-Jing Lee1,2, Amy Y M Au 3, Theodoros Foukakis1, Michela Barbaro1,Nimrod Kiss1, Roderick Clifton-Bligh3 , Johan Staaf 4 , Ake Borg4,Leigh Delbridge3, Bruce G Robinson3, Goran Wallin1, Anders Hoog 2

and Catharina Larsson1

1Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital, CMM L8:01, SE-17176

Stockholm, Sweden2Department of Oncology-Pathology, Karolinska Institutet, Karolinska University Hospital, SE-17176 Stockholm, Sweden3Kolling Institute of Medical Research, Royal North Shore Hospital, University of Sydney, Sydney, New South Wales, Australia4Department of Oncology, Lund University, Lund, Sweden

(Correspondence should be addressed to J-J Lee; Email: [email protected]; [email protected])

Abstract

Anaplastic thyroid cancer (ATC) is a rare but highly aggressive disease with largely unexplainedetiology andmolecular pathogenesis. In this study,weanalyzedgenome-wide copynumber changes,BRAF (V-raf sarcomaviral oncogenehomologB1)mutations, andp16 and cyclinD1expressions in apanel ofATCprimary tumors. ThreeATCsharbored thecommonBRAFmutationV600E.Usingarray-comparative genomic hybridisation (array-CGH), several distinct recurrent copy number alterationswere revealed including gains in 16p11.2, 20q11.2, and 20q13.12. Subsequent fluorescence in situhybridization revealed recurrent locus gain ofUBCH10 in 20q13.12 andCyclin D1 (CCND1) in 11q13.The detection of a homozygous loss encompassing the CDKN2A locus in 9p21.3 motivated theexamination of p16 protein expression, which was undetectable in 24/27 ATCs (89%). Based on thefrequent gain in 11q13 (41%;nZ11), the role ofCCND1was further investigated. Expression of cyclinD1 protein was observed at varying levels in 18/27 ATCs (67%). The effect of CCND1 on thyroid cellproliferation was assessed in vitro in ATC cells by means of siRNA and in thyroid cells after CCND1transfection. Insummary, the recurrent chromosomal copynumber changesandmolecular alterationsidentified in this study may provide an insight into the pathogenesis and development of ATC.


Introduction

Anaplastic thyroid cancer (ATC) is one of the most

aggressive human malignancies, with a median

survival of 3–6 months after diagnosis (Kondo et al.

2006). It is relatively rare comprising up to 5% of all

thyroid cancers and mainly affects the elderly (Kondo

et al. 2006). The natural history of the disease is

characterized by rapid and uncontrolled local growth

eventually causing suffocation. Distant metastases

frequently develop and are mainly located in the

lung. The treatment commonly involves radiotherapy

and chemotherapy that are given pre-operatively

followed by a surgery (Wallin et al. 2004).

Little is presently known about the cellular origin and

molecular etiology of ATC. This is partly attributed to

the extensive necrosis that is characteristic of the disease


1351–0088/08/015–801 q 2008 Society for Endocrinology Printed in Great

and further augmented by the pre-operative treatment.

In some patients, a differentiated thyroid cancer is found

adjacent to the ATC. Furthermore, in few cases, there is a

continuous spectrum from differentiated to poorly

differentiated thyroid cancer (PDTC) and ATC in support

of a progression model. TP53 mutations that are rare in

well-differentiated thyroid cancers, i.e., papillary thyroid

cancer (PTC) and follicular thyroid cancer (FTC), are

frequent in PDTC and reach up to 68% in ATC (Kondo

et al. 2006). PIK3CA mutations have been reported in

12–23% of ATC (Garcia-Rostan et al. 2005, Hou et al.

2007). Activating mutations of RAS (that are mainly

seen in FTC) and BRAF (V-raf sarcoma viral

oncogene homolog B1), which characterizes aggressive

PTC, are also found in a subset of ATCs while

RET (rearranged during transfection)/PTC and PAX8

Britain

DOI: 10.1677/ERC-08-0018

Online version via http://www.endocrinology-journals.org

http://dx.doi.org/10.1677/ERC-08-0018

J-J Lee et al.: Genetic alterations in anaplastic thyroid cancer

(paired box gene 8)/PPARg (peroxisome proliferator-

activated receptor-g) rearrangements have not been

determined in ATC (Kondo et al. 2006).

Studies of gene copy number imbalances in ATC

using conventional CGH have demonstrated recurrent

gains of chromosomal regions 3, 5p, 11q13, and 20q

and losses at 5q11–31 and Xp (Wreesmann et al. 2002,

Rodrigues et al. 2004). Recently, we characterized

karyotypic abnormalities and copy number alterations

in ATC cell lines, which revealed gain of 20q as the

most common abnormality (Lee et al. 2007). Here, we

used bacterial artificial chromosome (BAC) arrays

with whole-genome tiling resolution to investigate

the DNA copy number alterations in a series of

primary ATCs. Subsequently, we investigated the

involvement of candidate genes located in areas of

recurrent changes.

Table 1 Clinical information for the 28 cases of primary anaplastic

Follow-up

Case

no.

Sex

M/F

Age at

diagnosis

(yrs) Outcome Time

Preop.

therapy

Othe

tum

1 F 75 D 5.5 m No No

2 F 85 D 4.0 m Yes FTC

3 F 77 D 3.0 m Yes PTC

4 F 83 D 11.0 m Yes No

5 F 62 D 5.5 m Yes No

6 M 82 D 1.0 m Yes PTC

7 M 68 D 3.0 m Yes No

8 M 72 D 1.5 m Yes No

9 F 62 D 0.3 m Yes No

10 M 72 D 3.5 m Yes FTA

11 F 84 D 3.0 m Yes No

12 F 78 A 66.0 m Yes No

13 F 51 D 1.0 m Yes No

14 M 84 D 3.0 m Yes No

15 F 72 D 1.0 m Yes No

16 F 80 D 0.5 m Yes PTC

17 M 77 D 0.5 m No No

18 F 70 D 1.0 m Yes FTA

19 M 54 D 11.0 m Yes No

20 F 81 D 4.0 m Yes No

21 F 91 D 2.5 m Yes PDT

22 M 68 D 3.0 m Yes PTC

23 F 81 D 1.0 m Yes No

24 F 83 D 9.0 m Yes No

25 M 73 D 3.5 m Yes No

26 F 52 D 6.0 m No No

27 M 72 NA NA NA PTC

28 F 72 D 1.0 m NA No

F, female; M, male; yrs, years; m, months; NA, not available; D, dePTC, papillary thyroid cancer; FTA, follicular thyroid adenoma; PDT

802

Materials and methods

Established cell lines

The human ATC lines (HTh 104, HTh 112, HTh 7,

HTh 74, C 643, KAT-4, SW 1736, ARO, and HTh 83)

and Nthy-ori 3-1 (SV-40 immortalized normal human

thyroid follicular cells; ATCC, Manassas, VA, USA)

were cultured under conditions as described previously

(Lee et al. 2007).

Patients and tumor tissues

Fresh-frozen primary tumors from 28 cases of

ATC (Table 1) were collected at the Karolinska

University Hospital, Stockholm, Sweden and the Royal

North Shore Hospital, Sydney, Australia. Twenty-three

patients had received pre-operative radiotherapy and/or

chemotherapy according to standard treatment protocols

thyroid cancer (ATC) in the study

r thyroid

ors

Tumor

size

(cm)

Metastases/local

invasion at

diagnosis

Previous

goiter

8.5 No No

(cs) 4.0 No Yes

7.0 No Yes

6.0 No Yes

6.0 No No

4.0 NA No

5.0 Lung, lymph node Yes

10.0 Lung, lymph node,

local invasion

No

4.5 Lung No

6.5 Lung Yes

5.0 No No

8.5 No No

7.0 No No

10.0 No Yes

10.0 No Yes

8.0 No No

8.0 Lung Yes

5.0 No Yes

13.0 Local invasion No

5.0 No No

C (cs) 5.0 Lung, liver No

3.5 Lung, left adrenal,

local invasion

No

5.0 Lymph node No

3.5 No No

6.0 Lung, local invasion No

6.0 Local invasion No

6.5 Local invasion Yes

7.0 Lung Yes

ad; A, alive; Preop., preoperative; FTC, follicular thyroid cancer;C, poorly differentiated thyroid cancer; cs, continuous spectrum.

www.endocrinology-journals.org


(Wallin et al. 2004). The histopathological diagnosis was

established according to WHO classification (DeLellis

et al. 2004), including findings of undifferentiated cells,

giant and/or spindle cells, mitosis, and signs of necrosis.

Tissue sampling and representativity testing followed

established routines for the endocrine biobank. Frozen

samples of medullary thyroid cancer and parathyroid

adenoma were similarly collected at Karolinska

University Hospital, Stockholm, Sweden, and used as

references in western blot analyses. Informed consents

were obtained from all patients and ethical approvals

were granted.

DNA and protein extractions

Tumor DNA was isolated by conventional method-

ology including phenol purification and ethanol

precipitation. Cell line DNA was isolated as described

previously (Lee et al. 2007). DNA was quantified using

NanoDrop ND1000 (NanoDrop Technologies,

Wilmington, NC, USA).

BRAF mutation screening

The mutation hot spot exons 11 and 15 of BRAF were

sequenced on both strands in all 28 tumors. The

experimental procedure, amplification conditions (35

cycles), primers, and positive control were as pre-

viously described (Lee et al. 2007).

Array-CGH analysis

Generation, hybridization, and analyses of the 33 K

microarrays (resolution of 100 kb) with complete

genome coverage produced by the SCIBLU Genomics,

Department of Oncology, Lund University, Sweden

(http://www.lth.se/sciblu) were essentially as pre-

viously reported for 32 K arrays (Barbaro et al.

2007). Genomic DNA of the tumor and commercial

reference samples (Promega Corporation, Madison,

WI, USA) was labeled as described previously

(Jonsson et al. 2007). Arrays were scanned using

Axon GenePix 4200A microarray scanner (Molecular

Devices, Sunnyvale, CA, USA). Individual spots

identified on scanned arrays were collected using

GenePix Pro 6.0 (Axon Instruments, Foster City, CA,

USA), and the quantified data were loaded into Bio

Array Software Environment (BASE; Saal et al. 2002).

A BASE implementation of CGH-Plotter was used to

identify regions of gains and losses after smoothing

with a sliding window over three clones (Autio et al.

2003). Cut-off ratios for gains and losses constant were

set at Z1.15 and K0.87 respectively, corresponding to

log2 (ratio) of G0.2. A log2 (ratio) below K0.75 was


considered as homozygous loss and a ratio above

C0.75 as amplification.

Fluorescence in situ hybridization (FISH)

FISH analyses were performed on interphase imprints

from frozen ATCs and on metaphase preparations of

ATC cells using the BAC clone RP11-344G20 cover-

ing UBCH10 at 20q13.12 plus a chromosome 20

centromere probe (CEP20) as described previously

(Lee et al. 2007), or pre-labeled probes for CCND1 and

the chromosome 11 centromere (LSI CCND1 Spec-

trum Orange/CEP 11 SpectrumGreen1 Vysis, Inc.,

Downers Grove, IL, USA). Locus gain in a tumor was

considered when a higher number of signals were

recurrently observed for the gene-specific probe when

compared with the centromere probe. Results for

UBCH10 analysis of ATC cell lines have been

published in Lee et al. (2007).

Multiplex ligation-dependent probe amplification

(MLPA) analysis

Three regions with prominent gains detected from the

array, 20q11.2, 20q13.12, and 16p11.2, were selected

for verification by MLPA. MLPA reactions were

performed as described (Barbaro et al. 2007)

using newly designed 5 0 and 3 0 half-probes targeting

unique exonic or intronic sequences of genes within

20q11.2–q13.2 and 16p11.2 and control genes ALB

(4q13.3) and CLDN16 (3q28) according to Barbaro

et al. (2007) (Supplementary Table 1, which can be

viewed online at http://erc.endocrinology-journals.org/

supplemental/). For each sample, the peak areas

corresponding to each probe were first normalized to

the average of the peak areas of the control probes,

and then normalized to the average peak area in eight

controls (normal lymphocyte DNA).

Western blot analysis

Total protein extracts from tumor tissues (75 mg) and

cultured cell protein were electrophoresed and trans-

ferred to nitrocellulose filters (Invitrogen, Carlsbad,

CA, USA). For transfected cells, an aliquot was taken

from the cell suspension where the starting cell count

was 1!106 cells/well plated in a six-well plate. The

filters were stained with Ponceau Red (Sigma) as a

control for protein presence and incubated overnight at

4 8C with anti-cyclin D1 (1:400; SP4 clones; NeoMar-

kers, Fremont, CA, USA), anti-p16 (1:100; G175-405;

BD PharMingen, San Jose, CA, USA), and anti-a-

actinin (1:100; AT6/172 clone; Chemicon Inter-

national, Temecula, CA, USA) or anti-a-tubulin

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http://erc.endocrinology-journals.org/supplemental/



(1:2500; Clone DM 1A; Sigma–Aldrich). Anti-a-

actinin and anti-a-tubulin served as loading controls.

Cell proliferation analysis of cells

overexpressing CCND1

Amaxa nucleofection technology (Amaxa Biosystems,

Cologne, Germany) was used to transfect cells with

siRNA and plasmids for MTS assays. For cell

proliferation assays, 1!104 cells/well were plated

(96-well plate) and, for western blot analyses, 1!106

cells/well were plated (6-well plate). HTh 7 cells were

transfected with 1.5 mg siRNA/1!106 cells using

program X-001 with the V solution and Nthy-ori 3-1

cells were transfected with 2 mg plasmid/1!106 cells

using program A-020 with the T solution. Cells were

incubated for 16 h prior to subsequent analyses at 0, 24,

48, and 72 h after overnight transfection. CCND1

siRNA (#SI02654540, Qiagen GMbH, Valencia, CA,

USA) was used in knockdown studies with the All

Stars siRNA (#SI1027281, Qiagen) as negative

control. The CCND1 plasmid was obtained from

Addgene (Rc/CMV-CCND1 #8962, Cambridge, MA,

USA) and the control plasmid Rc/CMV was kindly

provided by Dr Sue Firth at the Kolling Institute of

Medical Research, NSW, Australia. Successful trans-

fection and siRNA were verified by western blot

analysis and quantitative real-time PCR (qRT-PCR) as

previously described (Lee et al. 2007). TaqMan Gene

Expression Assays (Applied Biosystems, Foster City,

CA, USA) were used to quantitate CCND1

(Hs00277039-m1) and 18S (#4319413E). Cyclin D1

expression from western blot analyses was quantified

against a-tubulin expression by Multi gauge V3.0

(FujiFilm Global, Valhalla, NY, USA).

Cell proliferation was quantitated by MTS assay

(Promega Corporation) as per protocol at 0, 24, 48, and

72 h after overnight transfection. Absorbance readings

(OD490–650) were taken 2 h after the addition of the

MTS reagent.

Statistical analysis

Potential correlations between the most fre-

quently altered regions (11q13, 20q11.2, 20q13.12,

13q21.2–q21.31, and 16p11.2) and CCND1 copy

number detected by FISH, cyclin D1 and p16

expressions, BRAF mutations, and association with

PTC were investigated using Fisher’s exact test (n!5)

or two-tailed c2-test (nO5), (http://www.graphpad.com/

quickcalcs/contingency1.cfm). For the validation of the

20q11.2, 20q13.12, and 16p11.2 regions identified from

the array by MLPA, f-correlation was computed

(Statistica version 6; StatSoft Inc., Johannesburg,

804

South Africa). Absorbance values of CCND1 siRNA

knockdown or CCND1-transfected cells were compared

with reference-treated cells using paired t-test. P values

below 0.05 were considered significant.

Results

Clinical characteristics and BRAF mutations

in primary ATCs

The clinical characteristics of the 28 cases of primary

ATCs studied are given in Table 1, and the molecular

analyses carried out in individual cases are detailed in

Table 2. Nine cases presented an additional thyroid

cancer that was either adjacent or growing in

continuous spectrum with the ATC. Three of the

twenty-eight ATCs exhibited a heterozygous nucleo-

tide alteration GTG/GAG at position 1799 in exon

15 of BRAF that leads to a missense mutation V600E

(Table 2). Two of these cases had an additional thyroid

cancer; case 6 exhibited a PTC adjacent to the ATC

and, in case 21, a continuous tumor spectrum from

PDTC to ATC was observed.

Array-CGH analysis of primary ATCs

DNA copy number changes were detected in all 27

primary ATCs successfully studied by array-CGH,

preferentially involving sub-chromosomal regions and

gains (Tables 2 and 3; Fig. 1). Alterations that were

commonly observed and further examined in this study

include gains at 11q13, 20q, and 16p11.2 (Figs 2, 3 and

4A). Other frequent events observed (O20% of cases)

include gains at 6p, 7q, 12q, 17q, 19, and 22q, and

losses on 4q and 13q. Gains in telomeres were

observed in O20% of the ATC panel for most

chromosomes with the exception of chromosomes 2,

3, 6, and 15.

The smallest regions of overlap (SRO) identified by

alignment of all alterations in individual tumors are

summarized in Table 3. Close to half of the panel

showed gain in 11q13 and the associated SRO at

11q12.2–q13.2 was altered in 33% of the tumors

(Table 3; Fig. 2A). Gain in 20q was one of the most

frequently observed aberrations (nZ14; Table 2),

where 52% of the panel showed gain in either 20q11.2

or 20q13.12 (Fig. 3A). Gain of 16p11.2 was observed

in 48% of the tumors (Fig. 4A; Table 3). In the long

arm of chromosome 22, gains were seen in the two

separate regions 22q11.21 (nZ16) and 22q13.1

(nZ15; Table 3). Similarly gain of chromosome 19

involved the 19p13 region (67% of the tumors) as well

as 19q13.1–q13.2 (52%). Gain in 7q11.22–q11.23 was

also recurrently observed in the panel (52%; Table 3).


http://www.graphpad.com/quickcalcs/contingency1.cfm

http://www.graphpad.com/quickcalcs/contingency1.cfm

Table 2 Molecular and genetic alterations of primary anaplastic thyroid cancer (ATC) detected in this study

Locus gain byFISH

Expressionby western

blot Gain by array-CGH within chromosome arm (sub-band) Array-CGH losses in

BRAFsequence

UBC-H10 CCND1

CyclinD1 p16 1q 6p 7q 11q 12q 16p 17q 19p 19q 20q 20q 22q 22q 4q 4q 13q

Caseno.

Otherthyroidtumors Ex 11C15 21 22–21 11.22–11.23 13 13 11.2 21 13 13.1–13.2 11.2 13.12 11.21 13.1 12–13.1 28.3 21.2–21.31

1 No wt Yes K C (K) K K K K K K K K K K K K K K K K2 FTC (cs) wt K K C (K) K K K K Yes K K K Yes K K Yes Yes K K K3 PTC wt Yes K CC (K) K K K K K Yes K Yes Yes Yes Yes K K K K Yes4 No wt Yes Yes CCC (K) Yes K Yes Yes Yes K Yes Yes K K Yes K K K K K5 No wt K Yes C (K) Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes K K K6 PTC V600E/wt Yes K CCC (K) K K K K K K K K Yes K K K K K Yes Yes7 No wt K Yes (K) (K) Yes Yes Yes Yes K Yes K Yes K K K K Yes K K K8 No wt Yes K CCC (K) K K K K Yes K K K K K K Yes Yes K K K9 No wt Yes Yes CCC (K) K K K Yes K K K K K Yes Yes Yes K K Yes K10 FTA wt K K (K) Yes K K K K K K K K Yes Yes K Yes K Yes Yes K11 No wt K K (K) (K) K K K K K K Yes K K K K K K K K K12 No wt K K CCC (K) K K K K Yes K Yes Yes K K K K K K K K13 No wt ND ND (K) (K) ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND14 No wt K Yes (K) (K) K Yes Yes Yes K Yes Yes Yes Yes K Yes Yes Yes K K K15 No V600E/wt K K CC Yes K K K K K K K Yes K K K K K K K K16 PTC wt K K CCC Yes K K Yes K Yes Yes K Yes K K K Yes Yes K Yes Yes17 No wt K K CCC (K) K K K K K K K K K K K K K K K K18 FTA wt K K C (K) K Yes Yes Yes Yes Yes Yes Yes Yes Yes K Yes Yes Yes Yes K19 No wt K K C (K) K K Yes K Yes Yes Yes Yes K K K Yes K Yes K Yes20 No wt K K CC (K) K K K K K Yes Yes Yes Yes K K K Yes K K K21 PDTC (cs) V600E/wt ND Yes (K) (K) K K Yes Yes Yes Yes Yes Yes Yes Yes K Yes Yes K K K22 PTC wt K Yes (K) (K) K K Yes K Yes Yes Yes Yes Yes Yes K Yes Yes Yes Yes K23 No wt K Yes CC (K) Yes K Yes Yes Yes Yes Yes Yes Yes Yes K Yes Yes Yes Yes K24 No wt Yes Yes CC (K) K Yes Yes Yes K Yes Yes Yes Yes Yes Yes Yes Yes K K K25 No wt ND ND (K) (K) Yes K Yes Yes Yes K Yes Yes K Yes Yes Yes Yes Yes Yes K26 No wt Yes K ND ND K K K K Yes Yes K Yes K Yes K K K K K K27 PTC wt ND Yes C (K) K Yes Yes Yes K K K Yes Yes Yes Yes Yes Yes Yes Yes Yes28 No wt Yes K (K) Yes K K Yes K K K K K Yes K K Yes Yes Yes K Yes

‘K’, Not present; Yes, present; ND, not determined; ‘(K)’, not detectable; ‘C’, low; ‘CC’, moderate; ‘CCC’, high; FTC, follicular thyroid cancer; PTC, papillary thyroid cancer; FTA,follicular thyroid adenoma; PDTC, poorly differentiated thyroid cancer; cs, continuous spectrum. Locus gain by FISH: UBCH10OCEP20 or CCND1OCEP11.

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Table 3 Frequently altered regions and associated smallest overlapping regions (SROs) in primary anaplastic thyroid cancers (ATCs)

Frequent alteration SROs

Cytoband

No. of cases

(Frequency) Cytoband bp start position (BAC) bp end position (BAC) Size (Mb)

No. of cases

(Frequency)

Copy number gains

C1q21 5 (19%) 1q21.1–q21.3 14 65 14 610 (RP11-437M17) 14 81 02 224 (RP11-787J14) 1.59 3 (11%)

C6p22–p21 6 (22%) 6p21.32 3 31 65 527 (RP11-521B19) 3 34 94 594 (RP13-512P23) 0.33 3 (11%)

C7q11.22–q11.23 14 (52%) 7q11.22–q11.23 7 20 35 816 (RP11-91L7) 7 22 59 198 (RP11-667P12) 0.22 13 (48%)

C11q13 11 (41%) 11q12.2–q13.2 6 33 21 142 (RP11-289J6) 6 62 12 707 (RP11-775M2) 2.89 9 (33%)

C12q13 13 (48%) 12q13.11–q13.12 4 73 84 342 (RP11-579D7) 4 86 39 263 (RP11-160B8) 1.25 8 (30%)

C16p11.2 13 (48%) 16p11.2 2 80 82 639 (RP11-410P5) 2 89 22 556 (RP11-674B7) 0.84 12 (44%)

C17q21 13 (48%) 17q21.33 4 59 49 240 (RP11-121F10) 4 64 66 966 (RP11-21I9) 0.52 8 (30%)

C19p13 18 (67%) 19p13.2 1 19 50 033 (RP11-566N12) 1 21 50 103 (RP11-754E16) 0.20 15 (56%)

C19q13.1–q13.2 14 (52%) 19q13.12 4 05 42 097 (RP11-166B11) 4 12 11 643 (RP11-532B13) 0.63 13 (48%)

C20q11.2 12 (44%) 20q11.21–q11.22 3 14 97 814 (RP11-120F10) 3 31 84 582 (RP11-612A10) 1.69 5 (19%)

C20q13.12 8 (30%) 20q13.12 4 18 87 694 (RP11-809G24) 4 41 53 298 (RP11-124K8) 2.27 8 (30%)

C22q11.21 16 (59%) 22q11.21 1 95 38 297 (RP11-54C2) 1 99 39 137 (RP11-818K20) 0.40 12 (44%)

C22q13.1 15 (56%) 22q13.1 3 60 03 989 (RP11-7I9) 3 64 44 264 (RP11-569A18) 0.44 13 (48%)

Copy number losses

K4q12–q13.1 8 (30%) 4q12–q13.1 6 13 17 501 (RP11-687A20) 6 60 95 641 (RP11-257M8) 4.78 8 (30%)

K4q28.3 9 (33%) 4q28.3 13 20 76 562 (CTD-2390L24) 13 31 23 563 (CTD-200F17) 1.05 6 (22%)

K13q21.2–q21.31 6 (22%) 13q21.2–q21.31 6 11 13 549 (RP11-418D23) 6 34 81 793 (RP13-495B8) 2.37 6 (22%)

J-J

Leeetal.:

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alte

ratio

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thyroid

cancer

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Figure 1 Frequency plot showing sequence copy number alterations detected in 27 primary ATCs for chromosomes 1–22. Gains aredepicted as green and losses as red vertical bars representing one ATC case each. Candidate genes and regions selected for furtheranalyses and the methods used are indicated below the plot.


Results from MLPA analysis (Supplementary Fig. 1,

which can be viewed online at http://erc.endocrinology-

journals.org/supplemental/) were concordant with gain

observed by array-CGH for 20q11.2 (fZ0.55),

20q13.12 (fZ0.51), and 16p11.2 (fZ0.54).

Copy number losses were most frequently observed in

4q12–q13.1 (30%), 4q28.3 (33%), and 13q21.2–q21.31

(22%; Table 3). Other recurrent alterations are gains at

1q21, 6p22–q21, 12q13, and 17q21 (Table 3). Interes-

tingly, ATC with concomitant PTC frequently showed

loss at 13q21.2–q21.31 (PZ0.003; Fisher’s test).

Comparison between genetic alterations with clinical

parameters (sex, age at diagnosis, survival, pre-

operative treatments, tumor size, metastasis, and

goiter) did not reveal additional statistically significant

correlations.

Amplifications and prominent losses revealed

by array-CGH in primary ATCs

Interestingly, high-level amplifications with log2

(ratio) exceeding C0.75 were identified in chromo-

somes 11, 18, and 20 for cases 7, 3, and 4 respectively.

ATC case 7 exhibited high-level amplification of a

5 Mb region in 11q22.1 (log2 (ratio)Z1.5–2.0;

Fig. 2A). Similarly, a 6 Mb region in 18q11.2 was

highly amplified in ATC case 3 (log2 (ratio)Z1.2;

Fig. 4B). Finally, in ATC case 4, high-level amplifi-

cation was observed of the commonly altered 20q13.12

region that includes the UBCH10 candidate gene

(Fig. 3A).

A homozygous loss of a 5 Mb region was detected in

ATC case 6 encompassing the CDKN2A gene locus in

9p21.3 (Fig. 4D). Furthermore, almost identical small

regional deletions within 5q were observed in cases

1 and 11, which included a common 1.9 Mb region in

5q13.2 (Fig. 4C). In these two cases, other regions


commonly altered in the ATC panel were largely

unaffected (Table 2).

Frequent lack of p16 expression in ATCs

Since a homozygous loss at the 9p21.3 locus

encompassing the CDKN2A gene was detected in

case 6, it was of interest to confirm the presence of

tumor suppressor p16 encoded by this locus for case

6 as well as to further investigate p16 expression in the

entire panel by western blot analysis. The p16 protein

expression was detected in positive control cells

SAOS-2 osteo sarcoma and in medullary thyroid

carcinoma tissue, but was not detectable in normal

thyroid or MCF-7 cells (negative control; Fig. 4D). In

addition, no p16 expression was observed in 24 out of

27 ATCs analyzed (89%; Table 2), including case 6

with homozygous loss at the CDKN2A locus.

Gains of chromosome 20 and locus gain

of UBCH10

Two separate regions of copy number gain were

observed for chromosome 20, of which the more distal

at 20q13.12 encompasses the UBCH10 gene that has

been suggested to be associated with ATC (Pallante

et al. 2005, Lee et al. 2007). We therefore performed a

dual-color FISH analysis on ATC imprints using a

CEP20 and a BAC clone containing UBCH10. Out of

24 samples, 9 (38%) showed an increased copy number

for UBCH10 (Table 2; Fig. 3B).

Gain of CCND1 in 11q13 and overexpression

of cyclin D1 protein

Amplification of chromosomal region 11q13 is

associated with gain for CCND1 in several human

cancers (Alao 2007). To determine whether the

CCND1 gene is gained in ATC tumors and cell lines,

807



Figure 2 (A) Array-CGH profiles of chromosome 11 for case 7 (upper) carrying amplification in 11q22.1 and gain in 11q13, and forcase 4 (below) harboring an 11q13 amplicon. (B) Western blot analyses showing cyclin D1 expression in positive control cells (C),and ATCs 21, 25, and 27, while normal thyroid (N) and ATCs 22, 23, and 24 are negative. Incubation of the same filter with a-actininserved as loading control. (C) Fluorescence in situ hybridization (FISH) of CCND1 (Cyclin D1, red) and centromere 11 (CEP11,green) copy numbers. Two signals are observed in normal metaphase and interphase nuclei, while ATC cases 4 and 7, and HTh 7cells show relative gain of CCND1.


dual-color FISH analysis was performed with a

CCND1 clone and centromere 11 (CEP 11) as a

control for chromosome copy number. As illustrated in

Fig. 2C, locus gain of CCND1 when compared with

CEP 11 was recurrently observed in interphase nuclei

of 38% of the ATCs (Table 2). The observation of

CCND1 locus gain coincides with the presence of

11q13 gain by array-CGH (PZ0.0001; two-tailed

c2-test). Locus gain of CCND1 was also recurrently

observed in interphase and metaphase cells of the ATC

line HTh 7 (Fig. 2C).

Western blot analysis showing strong cyclin D1

expression in parathyroid tumor tissue was used as a

positive control, while in normal thyroid cyclin D1

expression was not detectable (Fig. 2B). In primary

808

ATCs, varying levels of cyclin D1 expression were

observed in 18 out of 27 cases studied (Table 2).

Among these 18 ATCs, the protein level was

determined as low (six cases), intermediate (five

cases), or high (seven cases) as exemplified in Fig. 2B.

Effects of CCND1 on proliferation of thyroid cells

Gain of CCND1 or cyclin D1 overexpression was

observed in the majority of ATCs, while its possible

influence on thyroid cell proliferation was assessed in

ATC cells (HTh 7) and normal human thyroid cells

(Nthy-ori 3-1). The HTh 7 cells showed regional gain

of the CCND1 locus and overexpression of cyclin D1

protein and were therefore selected for transfection

with small interfering RNA (siRNA) oligonucleotides


Figure 3 (A) Array-CGH profiles of chromosome 20 for case 5 (upper), showing gain in 20q11.2 and 20q13.12, and case 4 (below)with high-level amplification in 20q13.12. (B) FISH analysis of UBCH10 (green) and centromere 20 (CEP20, red) in normalmetaphase and interphase nuclei, and in ATC cases 8, 9, and 3 with regional gain of UBCH10.


www.endocrinology-journals.org 809

Figure 4 (A) Array-CGH profile exemplifying the frequent gain of chromosome 16 in case 7, harboring a gain at 16p11.2. (B) Array-CGH profile of chromosome 18 for case 3 with amplification at the 18q11.2 region. (C) Array-CGH profile of chromosome 5,highlighting narrow deletion in 5q13.2 in case 1 (upper) and case 11 (below). (D) Array-CGH profile of chromosome 9 for case 6,harboring a homozygous loss at 9p21, where theCDKN2A gene is located. Western blot analyses show p16 expression in medullarythyroid cancer (MTC), ATC cases 10 and 28, and SAOS-2 cells used as a positive control (C). The p16 expression is not detected inMCF-7 cells (K), normal thyroid (N) or ATC cases 12, 11, 5, and 6. Subsequent incubation of the same filter with a-actinin served asloading control.


against CCND1. Successful siRNA within 24 h was

demonstrated by 20–30% decrease in western blot

expression and up to 60% decrease in CCND1 mRNA

expression by qRT-PCR (Fig. 5A). Slightly lower

proliferation measured by MTS assay absorbance was

observed after CCND1 siRNA when compared with

All Stars siRNA used as a control (Fig. 5). Transfection

of Nthy-ori 3-1 cells with a cyclin D1 expressing

construct resulted in stable 3-fold increase in protein

expression and 12-fold increase in mRNA levels

(Fig. 5B). Only minor increase in proliferation was

observed in cyclin D1 expressing cells when compared

810

with control cells transfected with empty vector

(Fig. 5). Taken together, CCND1 siRNA and transfec-

tion assays had only minor effects on proliferation,

which were not statistically significant.

Discussion

This is the first report of genome-wide detection of

DNA copy number changes in ATC using array-CGH.

A multitude of recurrent changes were detected in the

27 ATCs, and the role of candidate genes in selected


Figure 5 Analysis ofCCND1 (cyclin D1) effect on thyroid cell growth. Comparison plot for proliferation (MTS) assay (average of threeindependent experiments) of (A) HTh 7 ATC cells transfected against CCND1 siRNA and its reference control cells at 0, 24, 48, and72 h after transfection, and (B) Nthy-ori 3-1 normal thyroid cells transfected with CCND1 or empty vector (pRC/CMV). Standarderrors are indicated at each time point. Efficiency of siRNA as well asCCND1 transfection was validated by western blot analysis andqRT-PCR. Quantification was performed against a-tubulin for the western blot analysis and against 18S for qRT-PCR.


areas of chromosomal gain (CCND1 and UBCH10) or

loss (p16) was further examined.

An average of 44 DNA copy number changes was

detected in each tumor, which is considerably higher

than the changes found in differentiated thyroid cancer

(Hemmer et al. 1999, Kjellman et al. 2001, Wrees-

mann et al. 2002, 2004, Rodrigues et al. 2004). This

was rather expected, as aggressive and advanced

cancers are generally genetically unstable. The wide-

spread telomeric gains observed in this study is an

uncommon property of differentiated thyroid tumors,


further supporting chromosomal instability in ATC.

A role for telomere dysfunction in promoting gene

amplification and hence chromosome instability,

which is the hallmark of human cancer, is supported

by tumor-bearing mice model (Albertson 2006).

It could be argued that the observed copy number

alterations are a result of the pre-operative treatment

administered to the patients. However, the number and

patterns of changes in the three patients, which were

operated primarily without pre-treatment did not differ

from the rest. Furthermore, many of the alterations

811


were detected recurrently across the different tumors,

while alterations resulting from pre-operative radio-

chemotherapy are expected to be more random.

Gain of 11q13 was found by array-CGH and FISH

analysis for the CCND1 locus in w50% of ATCs. This

finding is in agreement with previous reports of 11q13

gain in ATC lines (Lee et al. 2007) and ATC primary

tumors (Wreesmann et al. 2002), and motivated further

investigation of the known oncogene CCND1 encoding

cyclin D1.

In protein studies, 67% of ATCs were shown to

express cyclin D1 while no expression was observed in

normal thyroid tissue. Cyclin D1 expression has been

reported to be especially prevalent in aggressive forms

of thyroid cancers (Wang et al. 2000, Khoo et al.

2002). Gain of 11q13 and/or CCND1 is also frequent in

other tumor types of relatively advanced stage,

including breast, head, and neck as well as esophagus

carcinomas, sometimes as part of homogenously

staining regions (Arnold & Papanikolaou 2005).

Notably, cyclin D1 overexpression in this study

occurred both in presence or absence of 11q13 gain,

suggesting alternative mechanisms of activation.

Similar observation has been previously reported in

breast cancers (Arnold & Papanikolaou 2005), kera-

toacanthoma (Burnworth et al. 2006), and squamous

cell carcinoma of the skin (Utikal et al. 2005). It has

also been proposed that in most cancer types,

pathogenic activation of cyclin D1 can occur via

additional mechanisms, including transcriptional and

post-transcriptional dysregulation by oncogenic sig-

nals (Arnold & Papanikolaou 2005). Consistent with

this possibility, in vitro experiments have shown direct

or indirect activation of the CCND1 promoter or cyclin

D1 expression by several molecules such as b-catenin,

c-Jun, PPARg, calveolin-1, Ras signaling, and others

(Arnold & Papanikolaou 2005). Intriguingly, three

putative microRNAs (miR-1, miR-206, and miR-613)

were predicted to target the 3 0 UTR of CCND1

(TargetScan 4.0), pointing to additional mechanisms

for regulation of cyclin D1 expression.

While cyclin D1 has been shown to promote cell

proliferation and drive tumorigenesis in several human

cancer models (Ewen & Lamb 2004), little is known

about its role in thyroid cancer. In this study,

introduction of cyclin D1 to normal thyroid cells

(Nthy-ori 3-1) resulted in an increased cell population

when compared with control cells. However, the

difference in growth rate did not reach statistical

significance. Unexpectedly, the population in HTh

7 cells transfected against CCND1 siRNA was only

marginally reduced when compared with cells without

CCND1 knockdown. This may be attributed to the

812

swift restoration of CCND1 within 24 h of transfection

against CCND1 siRNA. The rate of transcription and

translation of CCND1 within the cells of both in vitro

systems could vary, explaining the discrepancies

between cyclin D1 protein and CCND1 mRNA

expressions. The results from siRNA and overexpres-

sion of CCND1 suggest that cyclin D1 can stimulate

thyroid cell proliferation, but is in itself neither a

sufficient nor a necessary factor.

The identification of 20q11.2 and 20q13.12 ampli-

cons in this study corroborated our earlier findings in

ATC lines (Lee et al. 2007), as well as those reported in

ATC primary tumors (Wreesmann et al. 2002,

Rodrigues et al. 2004) and confirmed that the

amplicons of 20q are frequent events in ATC.

Interestingly, the only patient who was relapse free in

this study (case 12) did not exhibit 20q gain by array-

CGH. These findings suggest that 20q gain has a role in

the dedifferentiation of thyroid tumors. Recently,

overexpression of UBCH10, which resides in chromo-

somal region 20q13.12 and belongs to the E2 gene

family, was shown to be involved in thyroid cell

proliferation and was therefore suggested as a

candidate marker and possible therapy target for

ATC (Pallante et al. 2005). We observed locus gain

of UBCH10 in 25% of ATC tumors by FISH analysis,

which concurs with our previous observations in ATC

lines (Lee et al. 2007). Gain in 20q11.2–q21 and

20q13.12–q13.31 are also characteristics of other

human cancers (Hodgson et al. 2003, Weiss et al.

2003, Lockwood et al. 2007). Gain of 20q13.12 and

11q13 was recurrently found in the same ATC cases.

This could result from an unbalanced translocation

followed by an amplification event as observed in

lymphomas (Zhu et al. 2002), or reflect tumor

evolution with selection of clones amplifying growth-

promoting genes in different locations as reported in

breast cancers (Al-Kuraya et al. 2004). Translocations

in ATC have so far only been reported in ATC cell

lines, in particular involving chromosomes 11 (Lee

et al. 2007). CCND1 at the 11q13 locus is also known

to be frequently co-amplified with several other genes

at other chromosomes in breast carcinoma and head

neck and oral squamous cell cancers (Schuuring 1995).

Furthermore, co-amplification of CCND1 with genes

within the 11q13 cluster in oral squamous cancer has

been reported (Hsu et al. 2006).

A homozygous loss in the CDKN2 locus encoding

CDKN2A (p16INK4A) on chromosome 9p21 was

observed in one ATC, which was associated with

lack of p16 protein expression. This prompted us to

investigate p16 expression in the entire panel, which

revealed lack of p16 expression in 89% of the cases.



The lack of copy number loss at 9p21.3 in these ATCs

suggests other mechanisms for the inactivation of p16

such as methylation (Schagdarsurengin et al. 2006).

The normal thyroid tissues examined in our study did

not express p16. This observation is concurrent with

Ball et al. (2007) where the vast majority of normal

thyroid samples lacked p16 immunostaining. However,

we and others observed p16 expression in well-

differentiated thyroid tumors but not in ATC

(Fig. 4C; Ferru et al. 2006, Ball et al. 2007). Taken

together, these findings suggest that p16 is induced in

differentiated thyroid cancer and suppressed during

progression toward the undifferentiated phenotype.

Gain of 16p11.2 was frequently observed in this

study although this region encompasses no obvious

candidate oncogenes. However, this region was

identified as one of the most extensively duplicated

regions on chromosome 16 based on chromosome 16

genome sequencing (Martin et al. 2004). Loss of

chromosomal region 13q21 was exclusive to PTC-

associated ATCs (Table 3). Notably, recurrent loss of

13q21 has been reported in PTC (Kjellman et al. 2001,

Wreesmann et al. 2004). Conversely, loss of 4q

determined in our study has thus far only been

observed in ATC (Rodrigues et al. 2004, Lee et al.

2007). Restricted loss of 5q13.2 was noted in two

ATCs with low involvement of other recurrent

alterations. Of note, similar finding was previously

observed in an ATC line exhibiting concomitant

translocation of the 5q13 region (Lee et al. 2007).

Our results are consistent with previous works

(Wilkens et al. 2000, Miura et al. 2003, Pallante

et al. 2005, Lee et al. 2007) showing marked DNA

copy number alterations and frequent gains in ATCs;

suggesting high level of chromosomal instability in

ATC. Previous studies have shown that well-differ-

entiated tumors harbor fewer alterations (Hemmer

et al. 1999, Wreesmann et al. 2002). Three ATC

tumors harbored the common BRAF mutation V600E

that is frequently observed in PTC (Kondo et al. 2006).

The array-CGH profiling and BRAF mutation findings,

together with previous works, further support the

hypothesis previously suggested by Galera-Davidson

et al. (1987), that a subset of ATCs may be derived

from dedifferentiation of PTCs.

Taken together, DNA copy number changes were

found to be abundant in ATCs. Gains involving 20q

(20q11.2 and 20q13.12) and 11q13 represent recurrent

findings potentially targeting the candidate genes

CCND1/cyclin D1 and UBCH10. Lack of p16

expression and overexpression of cyclin D1 are

characteristics of ATCs, and cyclin D1 has a limited

effect on thyroid cell proliferation. The study revealed


several recurrent copy number alterations as well as

several candidate locations for tumor suppressor genes

and oncogenes that are potentially involved in

molecular pathogenesis of ATC.

Declaration of interest

The authors declare that there is no conflict of interest that

could be perceived as prejudicing the impartiality of the

research reported.

Funding

This study was supported by Swedish Cancer Society,

Swedish Research Council, Goran Gustafsson Foundation

for Research in Natural Sciences and Medicine, Gustav V

Jubilee Foundation, Cancer Society in Stockholm, Stockholm

County Council, and Swedish Medical Association. The

SCIBLU Genomics center is supported by grants from the K

& A Wallenberg Foundation and the Lund University.

Acknowledgements

We thank Prof. Lars Grimelius for the diagnostic work with

the thyroid tumors. We thank Dr Nils-Erik Heldin for

providing HTh7, HTh 73, C 643, SW 1736, HTh 104, HTh

83, and HTh 112, and Dr Kenneth Ain for providing KAT-4.

We also thank Ms Lisa Anfalk for excellent assistance in

collection of tumor samples.

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