Transformed diffuse large B-cell lymphomas with gains of thediscontinuous 12q12-14 amplicon display concurrentderegulation of CDK2, CDK4 and GADD153 genes

Follicular lymphoma (FL) accounts for 20–25% of all non-

Hodgkin lymphoma (NHL) (Harris et al, 1994; The Non-

Hodgkin’s Lymphoma Classification Project, 1997). FL is

characteristically associated with the chromosomal transloca-

tion t(14;18)(q32;q21), which juxtaposes the BCL-2 gene on

18q21 to the actively transcribed IGH gene on 14q32.

Consequently, the overproduction of BCL2 protein leads to

the inhibition of apoptosis (Godon et al, 2003). Although FL is

an indolent malignancy with a median survival of 7–8 years, it

is currently incurable. In addition, FL may undergo high grade

histological transformation to more aggressive malignancies,

most commonly diffuse large B-cell lymphoma (DLBCL) with

the frequency being highly variable (10–60%) (Bastion et al,

1997). Transformation is typically associated with the loss of

follicular histological architecture and acceleration of the

clinical course, with a loss of durable response to chemother-

apy and radiotherapy and a median survival less than 1 year

(Hubbard et al, 1982).

DLBCL constitutes 31% of all NHL types and is therefore

the most common (Harris et al, 1994; The Non-Hodgkin’s

Lymphoma Classification Project, 1997). In addition to some

of the cells retaining the t(14;18)(q32;q21) translocation

characteristic of FL (Richardson et al, 1987), the transformed

DLBCL cells also acquire multiple secondary genetic abnor-

malities at the cytogenetic (Armitage et al, 1988; Elenitoba-

Johnson et al, 1998; Cigudosa et al, 1999; Nanjangud et al,

2002) and molecular (Matolcsy et al, 1996; Pinyol et al, 1998;

Pasqualucci et al, 2001; Elenitoba-Johnson et al, 2003;

Osama Al-Assar,1 Karen S. Rees-

Unwin,1* Lia P. Menasce,2 Rachael

E. Hough,3 John R. Goepel,4 David

W. Hammond1 and Barry W. Hancock5

1Yorkshire Cancer Research Institute for Cancer

Studies, Division of Genomic Medicine, University

of Sheffield, Sheffield, 2Department of Pathology,

Christie Hospital, Manchester, 3Department of

Haematology, Sheffield Children’s Hospital,4Department of Pathology, Royal Hallamshire

Hospital, and 5Yorkshire Cancer Research

Academic Unit of Clinical Oncology, Cancer

Research Centre, Weston Park Hospital, Sheffield,

UK

Received 30 January 2006; accepted for

publication 7 March 2006

Correspondence: Dr O. Al-Assar, Institute for

Cancer Studies, Division of Genomic Medicine,

University of Sheffield, Sheffield S10 2RX, UK.

E-mail: o.al-assar@sheffield.ac.uk

*Present address: Dr Karen S. Rees-Unwin,

School of Life Sciences, The University of

Manchester, Jackson’s Mill, PO Box 88,

Manchester M13 OJH, UK.

Summary

Transformation of the indolent follicular lymphoma (FL) to the aggressive

diffuse large B-cell lymphoma (DLBCL) results in resistance to therapy with

shortened survival. It has been demonstrated that the 12q12-14 region was

mainly amplified in DLBCL cases but not in their FL counterparts. Therefore,

we examined the DNA copy number and protein expression profiles for

CDK2, CDK4 and GADD153, three genes that map to 12q12-14, in a set of

44 paired FL/DLBCL samples from 22 patients. The concordant amplification

of these genes occurred in seven of 22 (32%) of FL cases, compared with 15 of

22 (68%) of DLBCL cases. At the protein level, 15 of 22 of the DLBCL

samples (68%) showed strong staining for the CDK2 protein, compared with

five of 21 of FL samples (24%). The majority of the DLBCL samples (16/22,

72%) expressed the CDK4 protein, whereas the majority of the FL samples

(12/21, 57%) showed no expression of this protein. Except for one DLBCL

case, no expression of the GADD153 protein could be detected. The

deregulation of the CDK2 and CDK4 genes at the genetic and protein levels

suggest a functional role for these genes in the transformation process and

could potentially provide targets for prognostic tests or therapeutic

interventions.

Keywords: diffuse large B-cell lymphoma, follicular lymphoma, deregula-

tion, 12q12-14, genes.

research paper

ª 2006 The Authorsdoi:10.1111/j.1365-2141.2006.06093.x Journal Compilation ª 2006 Blackwell Publishing Ltd, British Journal of Haematology, 133, 612–621

Katzenberger et al, 2004) levels. These changes can involve

several genes but these do not appear to account for more than

a small number of cases (Lossos & Levy, 2003).

Previous work carried out by our group on a series of FL/

DLBCL paired lymphoma biopsies identified a region of

amplification on 12q12-14 that was detected in over half the

transformed DLBL cases and that was not seen in the FL

counterpart (Hough et al, 2001). These results suggested that

the 12q amplicon might be important in the transformation

from FL to DLBCL disease. The 12q12-14 amplicon is part of a

region that is associated with many malignancies (Riou et al,

1995; Reifenberger et al, 1996; Courjal & Theillet, 1997; Rao

et al, 1998). However, the amplification of 12q is highly

complex, exhibiting discontinuous regions of amplification

(Wolf et al, 1997) and regions that show gains or deletions do

not necessarily show conformity of amplification of genes

within these regions (Hyman et al, 2002; Martinez-Climent

et al, 2003; Zafarana et al, 2003). Importantly, a recent report

suggested that the 12cen-q15 amplicon is one of the regions

that could contain oncogenic pathways that are shared by the

various DLBCL subgroups (Bea et al, 2005). We, therefore,

investigated the amplification status and protein expression

profile of three selected genes that map to different regions on

the 12q12-14, as targets for the 12q12-14 amplification, after

recruiting additional samples that showed gains of the 12q12-

14 amplicon. These three genes were CDK2 (12q13.2), CDK4

(12q14.1) and GADD153 (12q13.3). In addition to being in

different regions on the 12q12-14, these genes were selected on

the basis of previous evidence of amplification and a role in cell

cycle (Rao et al, 1998; Maytin et al, 2001; Mao et al, 2002;

Murray, 2004).

Materials and methods

Clinical material

Paraffin embedded sections from paired historical biopsies of

22 patients with biopsy-proven FL at diagnosis and subsequent

transformation to DLBCL were obtained after being identified

using the regional clinical and pathological databases. This set

of paraffin embedded sections was used for DNA extraction

and included a previously published subset of tumour tissue

(Hough et al, 2001). The histological diagnosis was confirmed

prior to DNA extraction according to the World Health

Organisation classification (Harris et al, 1994).

Cell lines and paraffin embedding

The lymphoma cell line DoHH-2 (Kluin-Nelemans et al, 1991)

originated from a transformed lymphoma case and was

obtained from the German Collection of Micro-organisms

and Cell Culture. The lymphoma cell line HT (Beckwith et al,

1990) originated from a DLBCL case and was obtained from

the American Type Culture Collection (ATCC). The osteosar-

coma cell line SJSA-1 was obtained from the ATCC and was

selected because it showed amplification of the 12q using

comparative genomic hybridisation (CGH) (data not shown).

Paraffin embedding of cell lines was essentially as described by

others (Rhodes et al, 2002).

DNA extraction

Areas consisting of at least 50% tumour cells were identified by

immunocytochemistry using CD20, CD79a, CD43, CD3 and

CD45RO. Only sections with a similar percentage of tumour

cells were used for each FL/DLBCL pair. These areas were

micro-dissected from 6–10 serial tissue sections (5 mm) from

each biopsy. DNA was prepared using the Qiagen DNA tissue

kit and following the manufacturer’s protocol (Qiagen, UK).

DNA quality and integrity (OD260/OD280) was assessed using

spectophotometry (Lambda Bio UV/VIS spectrophotometer–

Perkin Elmer) and ethidium bromide stained Tris-buffered 1%

agarose gel.

Quantitative real time polymerase chain reaction

A total of 20–100 ng of extracted DNA was used in the

quantitative real time polymerase chain reaction (PCR)

reaction. A preliminary experiment has shown linear ampli-

fication of the template in the range 5–200 ng (data not

shown). PCR primers and TaqMan probe sequences (Table I)

were designed using the primer express 1.5 software against

published sequence-tagged site (STS) sequences and following

the Biosystems criteria. The probes were labelled with the

reporter dye 5,(6)-carboxyfluorescein at the 5¢ end and the

quencher dye 5,(6)-carboxy-tetramethyl-rhodamine at the 3¢

end. The reaction was performed using a PCR master mix with

5,(6)-carboxy-X-rhodamine as the background control dye

(Eurogentec, Oswell, UK), on a TaqMan ABI 7700 or 7900

(Applied Biosystems, Cheshire, UK). Control experiments

using identical samples were used to verify that both ABI 7700

Table I. Sequences of the primers and probes used in quantitative real-time polymerase chain reaction.

Gene Forward primer Reverse Primer TaqMan probe

Product

size (bp)

GAPDH TCTTACTCCTTGGAGGCCCATGT CATTTCCTGGTATGACAACGAATTT AATGAGGTCCACCACCCTGTTGCTGTAG 81

CDK2 CAGTTGCCAAGGATCCCTGAT GATGCCTAAACCCTAACTTCTAACTCCTA CCATTTTCCTCTGACGTCCACCTCCTACC 85

CDK4 GCTGCCATGGAAGGAAGAAA GCCTCAGAGATAAAGGCAAAGATT TGCCATTTCCCTTCTGGACACTGAGAG 77

GADD153 AAGTGGCTACTGACTACCCTCTCACTA GAGACCTTTCCTTTTGTCTACTCCAA TGACCCTCAATCCCACATACG CAGG 98

All sequences are in the 5¢–3¢ orientation.

CDK2, CDK4 and GADD153 deregulation in DLBCL

ª 2006 The AuthorsJournal Compilation ª 2006 Blackwell Publishing Ltd, British Journal of Haematology, 133, 612–621 613

and ABI 7900 yielded the same results. Primer and probe

concentrations were optimised for each gene (data not shown)

to give the lowest Ct and highest DRn for the same template

quantity, respectively. The PCR conditions used were as

follows: 2 min at 50�C, 10 min at 95�C and 40 cycles of 95�Cfor 15 s and 60�C for 1 min. The standard curves were

constructed within the linear range of amplification and using

serial dilutions of commercially available DNA pooled from

several individuals (Promega, Southampton, UK). This was

compared against standard curves constructed from DNA

pooled from 20 individuals in our laboratory and the results

were similar. All the samples were run in triplicates and the

plates were independently repeated in triplicates. The raw data

were analysed using sequence detection Software 2.0

(Applied Biosystems). Relative quantitation of the gene copy

number for each test gene was achieved by normalisation to

glyceraldehyde-3-phosphate dehydrogenase (GAPDH). All the

reactions were carried out as monoplex reactions as compe-

tition between the different sets of primers prevented multi-

plexing of the test gene with the endogenous gene in the same

reaction well (data not shown). A profile of normal gene copy

values for a specific gene was established from DNA extracted

from a panel of 20 normal individuals and included paraffin-

embedded normal tonsil sections. The normal range was

calculated by using the mean average of the 20 normal values

±2 SD. DNA extracted from formalin-fixed and paraffin-

embedded cell lines was used as a control. For statistical

analysis, one-tailed Student’s t-test for paired samples was used

to test the null hypothesis that the two sets of data came from

one population that has one mean, with the alternative

hypothesis being that one set had a larger mean than the other

(one tailed). The significance of the results was tested at an a of

0Æ05. The use of the Student’s t-test assumed normal distri-

bution of the population with standard deviation differences

being less than twofold between any two groups. Similar

skewness between the compared populations avoided distor-

tion of the P-values.

Immunohistochemistry

Five lm tissue sections mounted on 3-amino-propyl-tri-

ethoxysilone (APES)-coated slides were used for immuno-

staining. The sections were first de-paraffinised in xylene and

endogenous peroxidase activity was blocked using hydrogen

peroxide. Antigen retrieval was carried out by heating the

slides in a solution of 10 mmol/l tri-sodium citrate using a

microwave oven (800 W). The temperature was not allowed to

exceed 85�C to avoid disruption of tissue architecture and

maintain adhesion. The sections were then incubated in a

blocking solution of normal serum and incubated overnight at

4�C in the primary antibody solution. The CDK2 and CDK4

antibodies were obtained from Lab Vision/Neomarkers, Suf-

folk, UK) and the GADD153 antibody was obtained from

Santa Cruz Biotech (Middlesex, UK). For negative controls, a

solution containing IgG from the same primary antibody host

species was used (the same isotype). The immunoreactivity of

primary antibody was detected using a colorimetric method

and following the manufacturer’s instructions [Vectastain Elite

ABC and 3,3¢-diaminobenzidine (DAB) kits from Vector

Laboratories (Peterborough, UK)]. The immunostain images

were captured using a Nikon Cool Pix 990 digital camera

mounted on Nikon Biophot Microscope (Nikon UK Ltd,

Surrey, UK). The images were processed using nikonview 3TM

software. The evaluation of the staining of the sections

considered both the staining intensity and percentage of

positive tumour cells to take into consideration any hetero-

geneity of staining. The intensities of CDK2, CDK4 and

GADD153 expression were assessed by comparing the intensity

of the immunostain within the tumour cells to that of

surrounding ‘normal’ lymphoid cells, as determined by the

pathologists. In addition, the immunostaining results for a

particular candidate gene were compared with that of a

strongly positive (+3) control cell line. The three cell lines used

were DoHH-2, HT and SJSA-1 and they strongly stained for

CDK2, GADD153 and CDK4, except for DoHH-2, which

stained negatively for GADD153 and moderately (+2) for

CDK4. Using these criteria, tumours without any staining were

considered negative. Tumours with +1 staining intensity in

<80% of cells or +2 intensity in <30% of cells were considered

weakly positive (+1). Tumours with +1 staining intensity in

‡80% of cells, +2 intensity in 30–79%, or +3 intensity in <30%

were considered moderately positive (+2). Tumours with +2

intensity in ‡80%, or +3 intensity in ‡30% of cells were

considered strongly positive (+3). The correlation between the

quantitative real time PCR results and immunohistochemistry

was tested by Spearman’s s-test at an a of 0Æ05 (two-tailed),

using the Statistical Package for the Social Sciences (spss),

version 11 (SPSS Incorporation, Chicago, IL, USA).

Results

CDK2, CDK4 and GADD153 gene amplification

Figure 1 is a graphical representation of the gene amplification

results for the three genes investigated in this study. The gene

amplification results were normalised to the GAPDH gene. The

choice of an endogenous control gene localised to the 12 p arm

was due to the frequent occurrence of trisomy of chromosome

12 in lymphoma (Cabanillas et al, 1988; Offit et al, 1991).

Therefore, normalisation to the GAPDH gene rules out gene

copy number amplification due to entire chromosome 12 gain.

Normalisation to albumin resulted in comparable results (only

results showing normalisation to GAPDH are presented). The

normal ranges of CDK2, CDK4 and GADD153 gene copy

numbers in a series of 20 controls were 0Æ66–2Æ26, 0Æ31–2Æ20 and

0Æ38–1Æ96, respectively. Increased CDK2, CDK4 and GADD153

gene copy numbers were detected in 12 of 22 (55%), 12 of 22

(55%) and 18 of 22 (82%) of FL lymphoma cases, respectively.

In the transformed DLBCL lymphoma cases, increased CDK2,

CDK4 and GADD153 gene copy numbers were identified in 18

O. Al-Assar et al

ª 2006 The Authors614 Journal Compilation ª 2006 Blackwell Publishing Ltd, British Journal of Haematology, 133, 612–621

of 22 (82%), 20 of 22 (91%) and 20 of 22 (91%) of cases,

respectively. The concordant amplification of all three genes

occurred in seven of 22 (32%) of FL cases, compared with 15 of

22 (68%) of DLBCL cases. The average CDK2 gene copy

numbers were 2Æ70 (±0Æ23 SE) and 3Æ26 (±0Æ24 SE) in the FL

and transformed DLBCL lymphoma groups, respectively, with

the difference being statistically significant at a P-value of 0Æ002

(Fig 1A). No amplification of CDK2 was seen for any of the cell

lines examined. The average CDK4 gene copy numbers were

2Æ76 (±0Æ34 SE) and 4Æ14 (±0Æ42 SE) in the FL and DLBCL

groups, respectively, with the difference being statistically

significant (P ¼ 0Æ0009; Fig 1B). The SJSA-1 cell line showed a

23-fold increase in CDK4 gene copy number, whereas the

DoHH-2 and HT cell lines showed no amplification of this

gene. The average GADD153 gene copy numbers were 3Æ55

(±0Æ36 SE) and 4Æ23 (±0Æ33 SE) in the FL and DLBCL groups,

respectively, with the difference being statistically significant at

a P-value of 0Æ040 (Fig 1C). All the cell lines examined showed

GADD153 gene amplification, with the SJSA-1 cell line showing

the most prominent gain of 22 copies. In summary, the DLBCL

samples demonstrated the most prominent concurrent and

individual amplification of CDK2, CDK4 and GADD153 genes,

compared with the FL samples.

CDK2, CDK4 and GADD153 protein expression profiles

Table II details the scoring of the protein expression results for

the CDK2, CDK4 and GADD153 proteins. Immunostaining

for the CDK2 protein was detected in 20 of 21 cases (91%) and

all cases (100%) of the FL and DLBCL samples, respectively.

Some FL samples (8/21, 38%) showed moderate staining for

the CDK2 protein, whereas the majority of the DLBCL (15/22,

68%) samples showed strong staining. Figure 2A and B show

CDK2 staining in a representative case before and after

transformation, respectively. Immunostaining for the CDK4

protein was detected in nine of 21 cases (43%) and 16 of 22

cases (73%) of the FL and DLBCL cases, respectively. Most of

the FL samples (12/21, 57%) stained negatively for the CDK4

protein, whereas the majority of DLBCL samples (16/22, 73%)

stained positively for this protein. Figure 2C and D shows

CDK4 staining in a representative case before and after

transformation, respectively. Immunostaining for GADD153

protein could be detected in only one case (5%). Figure 2E and

F shows GADD153 staining in a representative case before and

after transformation, respectively. A graphical representation

of these results in the FL/DLBCL paired samples and a numeric

summary of these results presented as groups of FL and

DLBCL are shown in Fig 3 and Table III, respectively. There

was no statistically significant correlation between the gene

copy number and the protein expression in any of the groups.

Discussion

Areas of amplification and deletion identified in different

malignancies are good targets for the study of tumour

(A) CDK2

(C) GADD153

FL DLBCL

FL DLBCL

0

1

2

3

4

5

6

7

FL DLBCL

Gen

e co

py n

umbe

r

(B) CDK4

0

2

4

6

8

10

12

14

16

0

1

2

3

4

5

6

7

8

9

FL vs. DLBCLP-value = 0·002

FL vs. DLBCLP-value = 0·0009

FL vs. DLBCLP-value = 0·040

Fig 1. A graphical representation of the CDK2 (A), CDK4 (B) and

GADD153 (C) gene copy numbers in a series of 22 paired follicular

lymphoma/diffuse large B-cell lymphoma (FL/DLBCL) cases measured

by quantitative real-time polymerase chain reaction and normalised to

the GAPDH gene. The dotted lines describe the maximum values

detected in a panel of 20 normal controls and therefore provide a

threshold for positive gene amplification. The short, solid lines des-

cribe the mean average for that group. The P-values represent the

significance of the difference between the FL and DLBCL groups as

measured by the Student’s t-test.

CDK2, CDK4 and GADD153 deregulation in DLBCL

ª 2006 The AuthorsJournal Compilation ª 2006 Blackwell Publishing Ltd, British Journal of Haematology, 133, 612–621 615

development and progression. This is particularly important

when these changes are accompanied by changes in the protein

expression. It is assumed that chromosomal gains and

deletions have dosage effects on the expression of some of

the genes that map to these regions (Martinez-Climent et al,

2003). These changes in gene expression require high genomic

Table II. A summary of the protein expression

results for the CDK2, CDK4 and GADD153

proteins in follicular lymphoma/diffuse large B-

cell lymphoma (FL/DLBCL) cases and different

cell lines.

Total Number

Immunostaining strength

) +1 +2 +3

CDK2

FL 21 1 (5%) 7 (33%) 8 (38%) 5 (24%)

DLBCL

Cell lines

22

3

0 1 (5%) 6 (27%) 15 (68%)

SJSA-1, DoHH-2, HT

CDK4

FL 21 12 (57%) 9 (43%) 0 0

DLBCL

Cell lines

22

3

6 (27%) 9 (41%) 5 (23%)

DoHH-2

2 (9%)

SJSA-1, HT

GADD153

FL 21 0 0 0 0

DLBCL

Cell lines

22

3

0

DoHH-2

1 (5%) 0 0

SJSA-1, HT

Immunostaining strength is described as ‘)’ for negative expression, ‘+1’ for weak expression,

‘+2’ for moderate expression, and ‘+3’ for strong expression. The criteria for this categorisation

are described in the Materials and methods section.

Fig 2. Immunostaining for CDK2, CDK4 and GADD153 proteins in a representative paired lymphoma case using DAB substrate and haematoxylin

counterstain. (A) and (B) show CDK2 moderate (+2) and strong (+3) intensity staining in case 2 before and after transformation, respectively. (C)

and (D) show CDK4 weak (+1) and strong (+3) intensity staining in case 2 before and after transformation, respectively. (E) and (F) show GADD153

negative ()) and weak (+1) intensity staining in case 2 before and after transformation, respectively. Magnification was ·400 for all panels.

O. Al-Assar et al

ª 2006 The Authors616 Journal Compilation ª 2006 Blackwell Publishing Ltd, British Journal of Haematology, 133, 612–621

amplification, with the possibility of single (Monni et al, 1997)

or multiple (Kauraniemi et al, 2001) candidate genes mapping

to the same amplicon being involved. However, the presence of

a large number of target genes that map to the affected

chromosomal regions limits full exploration of potential

targets and the biological implications of these chromosomal

imbalances.

Therefore, to identify targets for the 12q12-14 amplification

in our samples, we examined the gene amplification status and

protein expression of three genes that map to different regions

on the 12q12-14: CDK2 (12q13.2), CDK4 (12q14.1) and

GADD153 (12q13.3). The choice of these genes does not

exclude the possibility of involvement of other genes in the

transformation process, especially in light of recent reports of

Patient number

N o r m a l R a n g eN o r m a l R a n g e

0

1

2

3

4

5

6

0123(A)

(B)

(C)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

Gen

e co

py n

umbe

rIH

C

CDK2DLBCL

FL

N o r m a l R a n g eN o r m a l R a n g e

IHC

0

1

2

3

4

5

6

7

8

9

15

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

0123

Gen

e co

py n

umbe

r

CDK4DLBCL

FL

N o r m a l R a n g eN o r m a l R a n g e

IHC

3

0

1

2

3

4

5

6

7

8

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

012

Gen

e co

py n

umbe

r GADD153DLBCL

FL

Fig 3. A graphical summary of the CDK2, CDK4 and GADD153 gene

amplification and protein expression results in all the samples pre-

sented as pairs of follicular lymphoma/diffuse large B-cell lymphoma

(FL/DLBCL). (A), (B) and (C) represent the CDK2, CDK4 and

GADD153 gene copy number and immunohistochemistry (IHC)

scoring, respectively. The shaded area on each graph represents the

normal range of the gene copy number for that gene.

Table

III.

Asu

mm

ary

of

the

gen

eam

pli

fica

tio

nan

dp

rote

inex

pre

ssio

nre

sult

so

fCDK2,

CDK4

andGADD153

info

llic

ula

rly

mp

ho

ma/

dif

fuse

larg

eB

-cel

lly

mp

ho

ma

(FL

/DL

BC

L)

case

s.

Gen

e

FL

DL

BC

L

P-V

alu

e

Agr

eem

ent

bet

wee

nge

ne

cop

yn

um

ber

and

pro

tein

exp

ress

ion

Agr

eem

ent

bet

wee

n

the

com

par

ativ

e

gen

om

ich

ybri

dis

atio

n

(CG

H)

resu

lts

and

gen

eco

py

nu

mb

er

Gen

eam

pli

fica

tio

n

(n¼

22)

Imm

un

oh

isto

chem

istr

y(n

¼21

)

Gen

eam

pli

fica

tio

n

(n¼

22)

Imm

un

oh

isto

chem

istr

y(n

¼22

)

Cas

esM

ean

±SE

)+

1+

2+

3C

ases

Mea

n±

SE)

+1

+2

+3

CDK2

12(5

5)2Æ

70±

0Æ23

1(5

)7

(33)

8(3

8)5

(24)

18(8

2)3Æ

26±

0Æ24

01

(5)

6(2

7)15

(68)

0Æ00

210

/21

(48)

6/22

(27)

CDK4

12(5

5)2Æ

76±

0Æ34

12(5

7)9

(43)

00

20(9

1)4Æ

14±

0Æ42

6(2

7)9

(41)

5(2

3)2

(9)

0Æ00

099/

21(4

3)8/

22(3

6)

GADD153

18(8

2)3Æ

55±

0Æ36

00

00

20(9

1)4Æ

23±

0Æ33

01(

5)0

00Æ

040

1/21

(5)

3/22

(14)

Th

eva

lues

inp

aren

thes

isar

egi

ven

asp

erce

nta

ges.

Th

ege

ne

amp

lifi

cati

on

resu

lts

are

rep

rese

nte

das

ap

erce

nta

geo

fca

ses

that

dem

on

stra

ted

ap

osi

tive

resu

lt(a

bo

veth

en

orm

alra

nge

)an

da

mea

n±

SEo

fth

eva

lues

for

all

the

case

s.

Th

eP

-val

ues

rep

rese

nt

the

stat

isti

cal

sign

ifica

nce

for

the

dif

fere

nce

bet

wee

nge

ne

amp

lifi

cati

on

resu

lts

inth

eF

Lversus

DL

BC

Lp

aire

dly

mp

ho

ma

sets

usi

ng

the

Stu

den

t’st-

test

asd

escr

ibed

inth

em

eth

od

s

sect

ion

.

Th

ere

sult

sfo

rth

eag

reem

ent

bet

wee

nth

ege

ne

cop

yn

um

ber

and

pro

tein

exp

ress

ion

use

dco

ncu

rren

tin

crea

ses

inb

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CDK2, CDK4 and GADD153 deregulation in DLBCL

ª 2006 The AuthorsJournal Compilation ª 2006 Blackwell Publishing Ltd, British Journal of Haematology, 133, 612–621 617

significant mRNA transcript increases of other genes, in

addition to CDK2 and CDK4, on the 12q12-14 amplicon in

DLBCL cases (Bea et al, 2005). In addition to previous reports

of the amplification of these genes in different malignancies

and role in the cell cycle (Rao et al, 1998; Mao et al, 2002; Bea

et al, 2005), these genes were selected because they showed no

amplification in a pilot study of a FL series with no evidence of

transformation to DLBCL (data not shown). Our published set

of paired FL/DLBCL samples was complemented with another

set of paired samples, which also showed amplification of the

12q12-14 amplicon in the transformed sample only using CGH

(data not shown). This provided us with a cohort of 44 paired

FL/DLBCL lymphoma samples from 22 patients.

A higher percentage of the DLBCL samples showed

amplification of the CDK2, CDK4 and GADD153 genes

compared with their FL counterparts (Fig 3 and Table III).

Furthermore, the concordant amplification of all these genes

occurred in 15 of 22 (68%) of DLBCL cases (Fig 3) compared

with only seven of 22 cases (32%) in the FL cases. Other

studies have demonstrated that CDK2 and CDK4 were both

deregulated at the genetic and mRNA levels (statistically

significant for CDK4 only in Bea et al, 2005) in DLBCL cases

(Martinez-Climent et al, 2003; Bea et al, 2005), and other

B-cell malignancies (Bea et al, 1999; Hernandez et al, 2005).

The level of CDK4 amplification in this study was also

demonstrated in a previous study (Rao et al, 1998). In

conclusion, the DLBCL samples in our study demonstrated

the most prominent concurrent and individual amplification

of CDK2, CDK4 and GADD153 genes, compared with the FL

samples.

Although these genes were concordantly amplified in seven

of 22 cases (32%) of FL cases, we could not detect an

amplification of the 12q12-14 in these cases. The inability of

CGH to detect amplifications in these seven FL cases is due

to its detection threshold, which is determined by the copy

number and the size of the amplified region (Bentz et al,

1998; Tachdjian et al, 2000). It is accepted that genetic

abnormalities are detected by CGH when the size of

chromosomal region affected is 10–12 Mb and smaller

regions can only be detected if there is a high level of

amplifications of target genes within these regions (Bentz

et al, 1998). Therefore, what appears to be a discrepancy

between the CGH and quantitative real time PCR results is

due to the intrinsic limitations of the former. We also verified

the amplification status of these three genes in one case using

interphase FISH (data not shown). The agreement between

the CGH results and gene copy numbers for the three genes

examined ranged between 14% and 36% of cases (3/22–8/22)

(Table III). Other researchers have also shown lack of

contiguity (Hyman et al, 2002; Zafarana et al, 2003) and

conformity of amplification of genes within regions of gains

or deletions detected in DLBCL cases by array CGH

(Martinez-Climent et al, 2003) or conventional CGH (Bea

et al, 2005). Because insufficient evidence has been provided

to link gene copy number changes and expression in the

transformation of B-cell NHL from FL to DLBCL, we

examined the protein expression of our candidate genes.

The CDK2 and CDK4 protein results should be considered

together because of their close link (reviewed by Sherr &

Roberts, 2004). The protein levels of these kinases are

biologically significant after binding to their cyclin partners

and becoming enzymatically active. Because CDK2 regulates

cell cycle progression through the interaction with both cyclin

E and cyclin A2, its functions extend beyond G1 regulation to

control events in S and G2/M phases. Early deregulation of the

CDK2 protein in our FL samples could have great impact on

the cell cycle with effects extending beyond G1, in light of the

broad substrates that CDK2 and its cyclin partners affect.

Unlike CDK2, the CDK4 protein showed different levels of

expression in the paired lymphoma samples (Fig 2C and D,

and Tables II and III). Although the majority of the FL cases

(57%, 12/21) showed no expression of the CDK4 protein, 73%

of DLBCL samples (16/22) expressed this protein. The

implications of deregulated CDK4 can be understood by

examining some of its biological functions. In complex with its

biological partner cyclin D, CDK4 has been shown to directly

affect the functions of CDK2 by mediating the expression of its

partners, cyclin E and cyclin A2, though E2F (Trimarchi &

Lees, 2002) and by sequestering p27Kip1 and p21Cip1, two of

CDK2 potent inhibitors (Sherr & Roberts, 2004). Therefore,

the deregulated expression of CDK4 in DLBCL could promote

further disruption of the cell cycle caused by an earlier CDK2

overexpression and aid in transformation. Essential for

oncogenic transformation could be the interplay among

CDK2, CDK4 and Cip/Kip proteins (Zou et al, 2002; Sherr

& Roberts, 2004). Other researchers have suggested that the

12cen-q15 amplicon might contain common oncogenic path-

ways for the different DLBCL subtypes (Bea et al, 2005).

Unfortunately, the effect of the deregulated expression of these

two kinases on the cell cycle in these tumours could not be

examined due to the lack of suitable biological material.

The CDK2 and CDK4 protein results were in agreement

with the dosage effect for these genes in 48% (10/21) and 43%

(9/21) of cases, respectively. In other malignancies too,

including other types of lymphomas, no absolute correlation

between genomic copy number and gene expression could be

demonstrated (Hughes et al, 2000; Phillips et al, 2001; Virta-

neva et al, 2001; Melendez et al, 2004), not even for highly

amplified genes (Hyman et al, 2002). Unfortunately, we could

only examine the mRNA levels in four patients for whom we

had suitable material. For those patients, there was an

agreement between the different genes copy numbers and

their mRNA transcripts (data not shown).

In contrast to the CDK2 and CDK4 profiles, the negative

expression of the GADD153 protein in all cases except one

demonstrated a complete lack of concordance with the relatively

high amplification levels of this gene (Fig 3C; Table III). The

mRNA for GADD153, which was examined in the limited

number of available samples using quantitative real time PCR

(data not shown), also showed increased transcript levels. Under

O. Al-Assar et al

ª 2006 The Authors618 Journal Compilation ª 2006 Blackwell Publishing Ltd, British Journal of Haematology, 133, 612–621

normal conditions, this ubiquitous early response gene is

expressed at low levels and its overexpression after exposure to

different agents has been shown to result in cell cycle arrest and

apoptosis (Maytin et al, 2001). Since lymphoma samples are

characterised by prolonged cell survival (McDonnell et al, 1990)

resulting from early overexpression of BCL-2, it is not surprising

that no expression of GADD153 could be detected in our

samples. The cases that we examined for the BCL-2 protein

showed strong staining for this protein (data not shown). It has

been demonstrated that overexpression of BCL-2 protein

suppresses GADD153-mediated apoptosis (McCullough et al,

2001). Furthermore, it has been shown that genes within the

same altered chromosomal regions respond differently to the

gain or loss of genetic material (Bea et al, 2005) and it has been

suggested that this is dependent on the individual regulation

mechanisms of these genes (Melendez et al, 2004). On the basis

of the mRNA results, these mechanisms could include post-

transcriptional control for GADD153 and emphasise a multi-

step transformation process (Martinez-Climent et al, 2003; de

Vos et al, 2003) that involves many alternative pathways.

In conclusion, this study provides the first report on the

status of CDK2, CDK4 and GADD153 proteins in pre- and

post-transformation B-cell lymphoma cases. The changes in

the gene dosage of these genes did not absolutely correlate with

increased protein levels. This is indicative of the involvement

of other gene specific regulatory mechanisms in controlling the

protein levels. In addition, these results highlight the import-

ance of examining the protein expression of candidate genes in

studying the transformation process to translate any gene

dosage changes into meaningful biological effects. The over-

expression of CDK2 in the vast majority of FL tumours could

entail a role for this kinase in oncogenesis and could possibly

complement apoptosis inhibition by the overexpression of the

BCL2 protein. The changes in CDK2 and CDK4 gene copy

numbers and protein overexpression in the majority of

transformed cases imply a functional role for these genes in

the multi-step transformation process mediated by their

multiple substrates and upstream targets. These targets effect

the biological functions of these kinases that are critical in

maintaining cell cycle regulation. Deregulation of CDK2 and

CDK4 would result in a perturbed cell cycle. In addition, these

two genes might be important targets for the 12q12-14

amplification in DLBCL but does not exclude the possibility

of involvement of other genes mapping to this region in the

transformation process. Other important genes on 12q12-14

should also be investigated for involvement in the transfor-

mation process. The lack of GADD153 protein expression in

almost all the samples highlights the involvement of factors

other than gene dosage underlying a complex transformation

process. These results could also help in identifying potential

targets for prognostic tests or therapeutic intervention. Iden-

tification of these targets could be greatly enhanced using a

functional proteomics approach to complement the profiles of

genomic aberrations and RNA expression in searching for

transformation-specific genes.

Acknowledgements

We thank David Edmundson, Yvonne Stephenson and Carrie

Wardle for their technical help in preparing the biopsy

material and paraffin embedding.

This work was funded by Weston Park Hospital Cancer

Appeal.

References

Armitage, J.O., Sanger, W.G., Weisenburger, D.D., Harrington, D.S.,

Linder, J., Bierman, P.J., Vose, J.M. & Purtilo, D.T. (1988) Correlation

of secondary cytogenetic abnormalities with histologic appearance in

non-Hodgkin’s lymphomas bearing t(14;18)(q32;q21). Journal of the

National Cancer Institute, 80, 576–580.

Bastion, Y., Sebban, C., Berger, F., Felman, P., Salles, G., Dumontet, C.,

Bryon, P.A. & Coiffier, B. (1997) Incidence, predictive factors, and

outcome of lymphoma transformation in follicular lymphoma

patients. Journal of Clinical Oncology, 15, 1587–1594.

Bea, S., Ribas, M., Hernandez, J.M., Bosch, F., Pinyol, M., Hernandez,

L., Garcia, J.L., Flores, T., Gonzalez, M., Lopez-Guillermo, A., Piris,

M.A., Cardesa, A., Montserrat, E., Miro, R. & Campo, E. (1999)

Increased number of chromosomal imbalances and high-level DNA

amplifications in mantle cell lymphoma are associated with blastoid

variants. Blood, 93, 4365–4374.

Bea, S., Zettl, A., Wright, G., Salaverria, I., Jehn, P., Moreno, V., Burek,

C., Ott, G., Puig, X., Yang, L., Lopez-Guillermo, A., Chan, W.C.,

Greiner, T.C., Weisenburger, D.D., Armitage, J.O., Gascoyne, R.D.,

Connors, J.M., Grogan, T.M., Braziel, R., Fisher, R.I., Smeland, E.B.,

Kvaloy, S., Holte, H., Delabie, J., Simon, R., Powell, J., Wilson,

W.H., Jaffe, E.S., Montserrat, E., Muller-Hermelink, H.K., Staudt,

L.M., Campo, E. & Rosenwald, A. (2005) Diffuse large B-cell lym-

phoma subgroups have distinct genetic profiles that influence tumor

biology and improve gene-expression-based survival prediction.

Blood, 106, 3183–3190.

Beckwith, M., Longo, D.L., O’Connell, C.D., Moratz, C.M. & Urba,

W.J. (1990) Phorbol ester-induced, cell-cycle-specific, growth

inhibition of human B-lymphoma cell lines. Journal of the National

Cancer Institute, 82, 501–509.

Bentz, M., Plesch, A., Stilgenbauer, S., Dohner, H. & Lichter, P. (1998)

Minimal sizes of deletions detected by comparative genomic

hybridization. Genes, Chromosomes and Cancer, 21, 172–175.

Cabanillas, F., Pathak, S., Trujillo, J., Manning, J., Katz, R.,

McLaughlin, P., Velasquez, W.S., Hagemeister, F.B., Goodacre, A. &

Cork, A. (1988) Frequent nonrandom chromosome abnormalities in

27 patients with untreated large cell lymphoma and immunoblastic

lymphoma. Cancer Research, 48, 5557–5564.

Cigudosa, J.C., Parsa, N.Z., Louie, D.C., Filippa, D.A., Jhanwar, S.C.,

Johansson, B., Mitelman, F. & Chaganti, R.S. (1999) Cytogenetic

analysis of 363 consecutively ascertained diffuse large B-cell lym-

phomas. Genes, Chromosomes and Cancer, 25, 123–133.

Courjal, F. & Theillet, C. (1997) Comparative genomic hybridization

analysis of breast tumors with predetermined profiles of DNA am-

plification. Cancer Research, 57, 4368–4377.

Elenitoba-Johnson, K.S., Gascoyne, R.D., Lim, M.S., Chhanabai, M.,

Jaffe, E.S. & Raffeld, M. (1998) Homozygous deletions at chromo-

some 9p21 involving p16 and p15 are associated with histologic

progression in follicle center lymphoma. Blood, 91, 4677–4685.

CDK2, CDK4 and GADD153 deregulation in DLBCL

ª 2006 The AuthorsJournal Compilation ª 2006 Blackwell Publishing Ltd, British Journal of Haematology, 133, 612–621 619

Elenitoba-Johnson, K.S., Jenson, S.D., Abbott, R.T., Palais, R.A.,

Bohling, S.D., Lin, Z., Tripp, S., Shami, P.J., Wang, L.Y., Coupland,

R.W., Buckstein, R., Perez-Ordonez, B., Perkins, S.L., Dube, I.D. &

Lim, M.S. (2003) Involvement of multiple signaling pathways in

follicular lymphoma transformation: p38-mitogen-activated protein

kinase as a target for therapy. Proceedings of the National Academy of

Sciences United States of America, 100, 7259–7264.

Godon, A., Moreau, A., Talmant, P., Baranger-Papot, L., Genevieve, F.,

Milpied, N., Zandecki, M. & Avet-Loiseau, H. (2003) Is

t(14;18)(q32;q21) a constant finding in follicular lymphoma? An

interphase FISH study on 63 patients. Leukemia, 17, 255–259.

Harris, N.L., Jaffe, E.S., Stein, H., Banks, P.M., Chan, J.K., Cleary, M.L.,

Delsol, G., De Wolf-Peeters, C., Falini, B., Gatter, K.C., Grogan,

T.M., Isaacson, P.G., Knowles, D.M., Mason, D.Y., Muller-Herme-

link, H.K., Pileri, S.A., Piris, M.A., Ralfkiaer, E. & Warnke, R.A.

(1994) A revised European-American classification of lymphoid

neoplasms: a proposal from the International Lymphoma Study

Group. Blood, 84, 1361–1392.

Hernandez, L., Bea, S., Pinyol, M., Ott, G., Katzenberger, T.,

Rosenwald, A., Bosch, F., Lopez-Guillermo, A., Delabie, J., Colomer,

D., Montserrat, E. & Campo, E. (2005) CDK4 and MDM2 gene

alterations mainly occur in highly proliferative and aggressive

mantle cell lymphomas with wild-type INK4a/ARF locus. Cancer

Research, 65, 2199–2206.

Hough, R.E., Goepel, J.R., Alcock, H.E., Hancock, B.W., Lorigan, P.C.

& Hammond, D.W. (2001) Copy number gain at 12q12-14 may be

important in the transformation from follicular lymphoma to dif-

fuse large B cell lymphoma. British Journal of Cancer, 84, 499–503.

Hubbard, S.M., Chabner, B.A., DeVita, Jr, V.T., Simon, R., Berard,

C.W., Jones, R.B., Garvin, A.J., Canellos, G.P., Osborne, C.K. &

Young, R.C. (1982) Histologic progression in non-Hodgkin’s lym-

phoma. Blood, 59, 258–264.

Hughes, T.R., Roberts, C.J., Dai, H., Jones, A.R., Meyer, M.R., Slade,

D., Burchard, J., Dow, S., Ward, T.R., Kidd, M.J., Friend, S.H. &

Marton, M.J. (2000) Widespread aneuploidy revealed by DNA mi-

croarray expression profiling. Nature Genetics, 25, 333–337.

Hyman, E., Kauraniemi, P., Hautaniemi, S., Wolf, M., Mousses, S.,

Rozenblum, E., Ringner, M., Sauter, G., Monni, O., Elkahloun, A.,

Kallioniemi, O.P. & Kallioniemi, A. (2002) Impact of DNA ampli-

fication on gene expression patterns in breast cancer. Cancer

Research, 62, 6240–6245.

Katzenberger, T., Ott, G., Klein, T., Kalla, J., Muller-Hermelink, H.K. &

Ott, M.M. (2004) Cytogenetic alterations affecting BCL6 are pre-

dominantly found in follicular lymphomas grade 3B with a diffuse

large B-cell component. American Journal of Pathology, 165, 481–490.

Kauraniemi, P., Barlund, M., Monni, O. & Kallioniemi, A. (2001) New

amplified and highly expressed genes discovered in the ERBB2

amplicon in breast cancer by cDNA microarrays. Cancer Research,

61, 8235–8240.

Kluin-Nelemans, H.C., Limpens, J., Meerabux, J., Beverstock, G.C.,

Jansen, J.H., de Jong, D. & Kluin, P.M. (1991) A new non-Hodgkin’s

B-cell line (DoHH2) with a chromosomal translocation

t(14;18)(q32;q21). Leukemia, 5, 221–224.

Lossos, I.S. & Levy, R. (2003) Higher grade transformation of follicular

lymphoma: phenotypic tumor progression associated with diverse

genetic lesions. Seminars in Cancer Biology, 13, 191–202.

Mao, X., Lillington, D., Child, F., Russell-Jones, R., Young, B. &

Whittaker, S. (2002) Comparative genomic hybridization analysis of

primary cutaneous B-cell lymphomas: identification of common

genomic alterations in disease pathogenesis. Genes, Chromosomes

and Cancer, 35, 144–155.

Martinez-Climent, J.A., Alizadeh, A.A., Segraves, R., Blesa, D., Rubio-

Moscardo, F., Albertson, D.G., Garcia-Conde, J., Dyer, M.J., Levy,

R., Pinkel, D. & Lossos, I.S. (2003) Transformation of follicular

lymphoma to diffuse large cell lymphoma is associated with a het-

erogeneous set of DNA copy number and gene expression altera-

tions. Blood, 101, 3109–3117.

Matolcsy, A., Casali, P., Warnke, R.A. & Knowles, D.M. (1996) Mor-

phologic transformation of follicular lymphoma is associated with

somatic mutation of the translocated Bcl-2 gene. Blood, 88, 3937–

3944.

Maytin, E.V., Ubeda, M., Lin, J.C. & Habener, J.F. (2001) Stress-in-

ducible transcription factor CHOP/gadd153 induces apoptosis in

mammalian cells via p38 kinase-dependent and -independent

mechanisms. Experimental Cell Research, 267, 193–204.

McCullough, K.D., Martindale, J.L., Klotz, L.O., Aw, T.Y. & Holbrook,

N.J. (2001) Gadd153 sensitizes cells to endoplasmic reticulum stress

by down-regulating Bcl2 and perturbing the cellular redox state.

Molecular and Cellular Biology, 21, 1249–1259.

McDonnell, T.J., Nunez, G., Platt, F.M., Hockenberry, D., London, L.,

McKearn, J.P. & Korsmeyer, S.J. (1990) Deregulated Bcl-2-im-

munoglobulin transgene expands a resting but responsive

immunoglobulin M and D-expressing B-cell population. Molecular

and Cellular Biology, 10, 1901–1907.

Melendez, B., Diaz-Uriarte, R., Cuadros, M., Martinez-Ramirez, A.,

Fernandez-Piqueras, J., Dopazo, A., Cigudosa, J.C., Rivas, C.,

Dopazo, J., Martinez-Delgado, B. & Benitez, J. (2004) Gene

expression analysis of chromosomal regions with gain or loss of

genetic material detected by comparative genomic hybridization.

Genes, Chromosomes and Cancer, 41, 353–365.

Monni, O., Joensuu, H., Franssila, K., Klefstrom, J., Alitalo, K. &

Knuutila, S. (1997) BCL2 overexpression associated with chromo-

somal amplification in diffuse large B-cell lymphoma. Blood, 90,

1168–1174.

Murray, A.W. (2004) Recycling the cell cycle: cyclins revisited. Cell,

116, 221–234.

Nanjangud, G., Rao, P.H., Hegde, A., Teruya-Feldstein, J., Donnelly,

G., Qin, J., Jhanwar, S.C., Zelenetz, A.D. & Chaganti, R.S. (2002)

Spectral karyotyping identifies new rearrangements, translocations,

and clinical associations in diffuse large B-cell lymphoma. Blood, 99,

2554–2561.

Offit, K., Jhanwar, S.C., Ladanyi, M., Filippa, D.A. & Chaganti, R.S.

(1991) Cytogenetic analysis of 434 consecutively ascertained speci-

mens of non-Hodgkin’s lymphoma: correlations between recurrent

aberrations, histology, and exposure to cytotoxic treatment. Genes,

Chromosomes and Cancer, 3, 189–201.

Pasqualucci, L., Neumeister, P., Goossens, T., Nanjangud, G., Chag-

anti, R.S., Kuppers, R. & Dalla-Favera, R. (2001) Hypermutation of

multiple proto-oncogenes in B-cell diffuse large-cell lymphomas.

Nature, 412, 341–346.

Phillips, J.L., Hayward, S.W., Wang, Y., Vasselli, J., Pavlovich, C.,

Padilla-Nash, H., Pezullo, J.R., Ghadimi, B.M., Grossfeld, G.D.,

Rivera, A., Linehan, W.M., Cunha, G.R. & Ried, T. (2001) The

consequences of chromosomal aneuploidy on gene expression pro-

files in a cell line model for prostate carcinogenesis. Cancer Research,

61, 8143–8149.

Pinyol, M., Cobo, F., Bea, S., Jares, P., Nayach, I., Fernandez, P.L.,

Montserrat, E., Cardesa, A. & Campo, E. (1998) p16(INK4a) gene

O. Al-Assar et al

ª 2006 The Authors620 Journal Compilation ª 2006 Blackwell Publishing Ltd, British Journal of Haematology, 133, 612–621

inactivation by deletions, mutations, and hypermethylation is

associated with transformed and aggressive variants of non-Hodgkin’s

lymphomas. Blood, 91, 2977–2984.

Rao, P.H., Houldsworth, J., Dyomina, K., Parsa, N.Z., Cigudosa, J.C.,

Louie, D.C., Popplewell, L., Offit, K., Jhanwar, S.C. & Chaganti, R.S.

(1998) Chromosomal and gene amplification in diffuse large B-cell

lymphoma. Blood, 92, 234–240.

Reifenberger, G., Ichimura, K., Reifenberger, J., Elkahloun, A.G.,

Meltzer, P.S. & Collins, V.P. (1996) Refined mapping of 12q13-q15

amplicons in human malignant gliomas suggests CDK4/SAS and

MDM2 as independent amplification targets. Cancer Research, 56,

5141–5145.

Rhodes, A., Jasani, B., Couturier, J., McKinley, M.J., Morgan, J.M.,

Dodson, A.R., Navabi, H., Miller, K.D. & Balaton, A.J. (2002) A

formalin-fixed, paraffin-processed cell line standard for quality

control of immunohistochemical assay of HER-2/neu expression in

breast cancer. American Journal of Clinical Pathology, 117, 81–89.

Richardson, M.E., Chen, Q.G., Filippa, D.A., Offit, K., Hampton, A.,

Koduru, P.R., Jhanwar, S.C., Lieberman, P.H., Clarkson, B.D. &

Chaganti, R.S. (1987) Intermediate-to high-grade histology of

lymphomas carrying t(14;18) is associated with additional nonran-

dom chromosome changes. Blood, 70, 444–447.

Riou, G., Barrois, M., Prost, S., Terrier, M.J., Theodore, C. & Levine,

A.J. (1995) The p53 and mdm-2 genes in human testicular germ-cell

tumors. Molecular Carcinogenesis, 12, 124–131.

Sherr, C.J. & Roberts, J.M. (2004) Living with or without cyclins and

cyclin-dependent kinases. Genes and Development, 18, 2699–2711.

Tachdjian, G., Aboura, A., Lapierre, J.M. & Viguie, F. (2000) Cyto-

genetic analysis from DNA by comparative genomic hybridization.

Annales de genetique, 43, 147–154.

The Non-Hodgkin’s Lymphoma Classification Project (1997) A clinical

evaluation of the International Lymphoma Study Group classifica-

tion of non-Hodgkin’s lymphoma. The Non-Hodgkin’s Lymphoma

Classification Project. Blood, 89, 3909–3918.

Trimarchi, J.M. & Lees, J.A. (2002) Sibling rivalry in the E2F family.

Nature Reviews Molecular Cell Biology, 3, 11–20.

Virtaneva, K., Wright, F.A., Tanner, S.M., Yuan, B., Lemon, W.J.,

Caligiuri, M.A., Bloomfield, C.D., de La Chapelle, A. & Krahe, R.

(2001) Expression profiling reveals fundamental biological differ-

ences in acute myeloid leukemia with isolated trisomy 8 and normal

cytogenetics. Proceedings of the National Academy of Sciences United

States of America, 98, 1124–1129.

de Vos, S., Hofmann, W.K., Grogan, T.M., Krug, U., Schrage, M.,

Miller, T.P., Braun, J.G., Wachsman, W., Koeffler, H.P. & Said, J.W.

(2003) Gene expression profile of serial samples of transformed

B-cell lymphomas. Laboratory Investigation, 83, 271–285.

Wolf, M., Aaltonen, L.A., Szymanska, J., Tarkkanen, M., Blomqvist, C.,

Berner, J.M., Myklebost, O. & Knuutila, S. (1997) Complexity of

12q13–22 amplicon in liposarcoma: microsatellite repeat analysis.

Genes, Chromosomes and Cancer, 18, 66–70.

Zafarana, G., Grygalewicz, B., Gillis, A.J., Vissers, L.E., van de Vliet, W.,

van Gurp, R.J., Stoop, H., Debiec-Rychter, M., Oosterhuis, J.W., van

Kessel, A.G., Schoenmakers, E.F., Looijenga, L.H. & Veltman, J.A.

(2003) 12p-amplicon structure analysis in testicular germ cell

tumors of adolescents and adults by array CGH. Oncogene, 22,

7695–7701.

Zou, X., Ray, D., Aziyu, A., Christov, K., Boiko, A.D., Gudkov, A.V. &

Kiyokawa, H. (2002) Cdk4 disruption renders primary mouse cells

resistant to oncogenic transformation, leading to Arf/p53-in-

dependent senescence. Genes and Development, 16, 2923–2934.

CDK2, CDK4 and GADD153 deregulation in DLBCL

ª 2006 The AuthorsJournal Compilation ª 2006 Blackwell Publishing Ltd, British Journal of Haematology, 133, 612–621 621