Original paper
b-catenin nuclear expression correlates with cyclin D1overexpression in sporadic desmoid tumours
Tsuyoshi Saito1, Yoshinao Oda1, Kazuhiro Tanaka2, Shuichi Matsuda2, Sadafumi Tamiya1, Yukihide Iwamoto2
and Masazumi Tsuneyoshi1*1 Department of Anatomic Pathology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan2 Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
*Correspondence to:Masazumi Tsuneyoshi,Department of AnatomicPathology (Second Departmentof Pathology), PathologicalSciences, Graduate School ofMedical Sciences, KyushuUniversity, Maidashi 3-1-1,Higashi-ku, Fukuoka 812-8582,Japan.E-mail: [email protected]
Received: 13 December 2000
Revised: 31 January 2001
Accepted: 3 April 2001
Published online: 8 August 2001
Abstract
The immunohistochemical expression of b-catenin, cyclin D1, Ki-67 and PCNA was Examined in
38 cases of sporadic extra-abdominal or abdominal-wall desmoid tumours without familial
adenomatous polyposis (FAP), to evaluate the hypothesis that the accumulated b-catenin within
the nuclei could affect the regulation of the cyclin D1 gene. There was a statistically significant
correlation between b-catenin accumulation and cyclin D1 overexpression ( p=0.029). Each group
with b-catenin accumulation or cyclin D1 overexpression showed a higher PCNA-LI than those
without, the difference being statistically significant (p=0.007, p=0.004, respectively).
Differential PCR was also performed to detect amplification of the cyclin D1 gene and
mutational analysis was undertaken for exon 3 of the b-catenin gene. Amplification of the cyclin
D1 gene was observed in 13 out of 22 cases (59.1%). There were nine-point mutations in 7 out of
18 cases (38.9%). The distribution of b-catenin mutation fell within a wide range, from codon 21
to codon 67. In conclusion, b-catenin nuclear expression correlated with cyclin D1 overexpression
in sporadic desmoid tumours, which could be an in vivo model system for the APC-b-catenin-Tcf
pathway. In addition, b-catenin mutations in desmoid tumours occurred at an unusually wide
range of sites within the gene. Copyright # 2001 John Wiley & Sons, Ltd.
Keywords: desmoid tumour; b-catenin; mutation; cyclin D1; Ki-67; PCNA
Introduction
It has been reported that b-catenin is a multifunctional
protein involved in the wingless/Wnt signal transduc-
tion pathway, in addition to being a cell–cell adhesion
regulator when binding to E-cadherin adhesion mole-
cules [1,2]. The binding of b-catenin to adenomatous
polyposis coli (APC) protein requires phosphorylation
of b-catenin by GSK (glycogen synthase kinase)-3b on
serine/threonine residues, all of which are encoded in
exon 3 of the gene. In colorectal cancers, mutations of
APC or b-catenin result in stabilization of b-catenin
and a significant accumulation of this protein within
the cytoplasm [3]. Furthermore, increased b-catenin
may translocate to the nuclei and could serve as a
transcriptional factor by binding to the T-cell factor/
lymphoid enhancing factor (Tcf–Lef) family [3]. More-
over, we recently demonstrated that the accumulated
b-catenin within the nuclei of tumour cells served as an
oncoprotein in synovial sarcoma, increasing its pro-
liferative activity as assessed by Ki-67, thus resulting in
a poor overall survival rate [4]. However, it was still
not clear how the accumulated b-catenin within the
nuclei could act as an oncoprotein in the APC-b-
catenin-Tcf pathway.Desmoid tumour is an infiltrative and locally
aggressive lesion characterized by florid fibroblastic
proliferation. Desmoid tumours are also known to
have a high frequency of APC and b-catenin mutations
[5–9]. Li et al. [10] demonstrated that APC truncating
mutations provide aggressive fibromatosis cells with a
proliferative advantage through b-catenin and sug-
gested that b-catenin acted to transactivate transcrip-
tion. Desmoid tumors could therefore be thought of as
providing an in vivo model system for the APC-b-
catenin-Tcf pathway.Recently, some of the targeted genes of the APC-b-
catenin-Tcf pathway have been identified [11–14]; one
of the candidates is thought to be the cyclin D1 gene
[14]. Indeed, more recently, Tetsu et al. [14] demon-
strated that abnormal levels of b-catenin may con-
tribute to neoplastic transformation by causing the
accumulation of cyclin D1 in colon carcinoma cells.
However, it remains unclear whether b-catenin accu-
mulation could affect the regulation of the cyclin D1
gene in vivo.In this study, to test the hypothesis that the
accumulated b-catenin within the nuclei could affect
the regulation of the cyclin D1 gene, we examined the
immunohistochemical expression of b-catenin, cyclin
D1, Ki-67 and PCNA in sporadic extra-abdominal or
abdominal-wall desmoid tumours without familial
adenomatous polyposis (FAP), combined with some
molecular analyses.
Journal of PathologyJ Pathol 2001; 195: 222–228.DOI: 10.1002 /path.942
Copyright # 2001 John Wiley & Sons, Ltd.
Materials and methods
Materials
Thirty-eight formalin-fixed, paraffin-embedded cases ofsporadic (28 extra-abdominal and 10 of abdominal-wall) desmoid tumours without FAP were selectedfrom among the collection of soft-tissue tumoursregistered in the Department of Anatomic Pathology,Pathological Sciences, Graduate School of MedicalSciences Kyushu University, Japan.
Immunohistochemistry
Immunohistochemistry was performed using anti-b-catenin mouse monoclonal antibody (mAb) (clone 14,Transduction Laboratories, Lexington KY; 1 : 200),anti-cyclin D1 mouse mAb (clone P2D11F11,Novocastra; 1 : 25), anti-PCNA mouse mAb (clonePC10, Dakopatts, Copenhagen, Denmark; 1 : 100) andanti-Ki-67 mouse mAb (clone MIB-1, Immunotech,Marseille, France; 1 : 100). For evaluating b-cateninstaining, sections were considered to demonstratewidespread nuclear staining if more than 75% of thetumour cells showed nuclear staining [4,15]. Forevaluating the immunohistochemistry of cyclin D1,sections were considered as demonstrating over-expression if more than 5% of the cells were positivelystained [16–18]. The MIB-1-labelling index (LI) andPCNA-LI were expressed as previously described [4].
DNA extraction
Genomic DNA was purified from formalin-fixedparaffin-embedded material as previously described[4]. Genomic DNA was also extracted, using a DNAextraction kit, Sepa Gene, from an HeLa cell linepreviously described as lacking any amplification of thecyclin D1 gene, as a standard positive control fordifferential PCR [19].
Differential PCR
The primer sequences for the cyclin D1 gene and thedopamine-D2-receptor gene, used as an internal con-trol, have been previously described [20]. The PCRprotocol included initial denaturing at 96uC for fiveminutes, followed by 40 cycles of one minute each at96uC, 50uC and 72uC, and after the final cycle ofamplification, the extension was continued for anadditional seven minutes at 72uC. To avoid any lossof assay accuracy and to determine the appropriatenumber of PCR cycles, different cycles of PCRamplification were performed using DNA from HeLacells. The cyclin D1/dopamine receptor ratios werealmost constant at 35 to 40 cycles, so all clinicalspecimens were analysed using 40 cycles. Followingamplification, the products were electrophoresedthrough 3.0% agarose gel with ethidium bromide at100 V for 50 minutes. The UV-illuminated gels werephotographed using Polaroid film. The intensities ofthe DNA products were quantified using a National
Institutes of Health (NIH) image version 1.56. Thelevel of cyclin D1 amplification was determined bycomparing the ratio of the intensities of the cyclin D1and dopamine-D2-receptor PCR products for each ofthe samples with positive HeLa cells. Samples showingmore than two-fold amplification were judged asamplified.
Polymerase chain reaction–single-strandconformation polymorphism (PCR-SSCP) andmutational analysis of b-catenin gene
A genomic PCR fragment including the entire regionof exon 3 was amplified using a previously describedpair of intronic primers at 58uC of annealing tempera-ture [21]. Human genomic DNA (CLONTECH) wasused as a positive control for each PCR and for thesubsequent reactions. SSCP was performed as pre-viously described [4]. To increase the quantity ofmutant DNA prior to sequencing, extra bands whichseemed to be aberrantly migrating were excised fromthe SSCP gel, and re-amplified for 25 cycles under thesame conditions. The samples were analysed forsequencing after the subsequent reaction.
Statistical analysis
Fisher’s exact test was performed to assess thecorrelation among the various immunohistochemicalresults. A p-value <0.05 was considered significant.
Results
Immunohistochemistry
In all cases, positive staining for b-catenin wasrecognized in the nuclei and cytoplasm of the fibro-blastic tumour cells; b-catenin-positive cells appearedto be uniformly distributed throughout the sections,although the proportion of positive cells varied(Figures 1A, 2A). Nineteen cases (50%) were judgedas showing widespread nuclear staining. There was nostatistically significant correlation between the wide-spread nuclear staining of b-catenin and the tumourlocation (abdominal wall or extra-abdominal). On theother hand, cyclin D1-positive cells also appeared to beevenly scattered throughout the section (Figures 1B,2B), and 27 cases (71.1%) showed overexpression ofcyclin D1. Among 19 cases with widespread nuclearstaining of b-catenin, 17 showed overexpression ofcyclin D1. The remaining two cases were abdominal-wall desmoid tumours. Extra-abdominal desmoidtumours had a tendency to show cyclin D1 over-expression more frequently than abdominal-wall des-moid tumours, although this was not statisticallysignificant ( p=0.08). There was a statistically sign-ificant correlation between widespread nuclear stainingof b-catenin and cyclin D1 overexpression ( p=0.029,Table 1). With the exception of a small number ofcases, Ki-67-positive tumour cells either could not bedetected, or else were very few (Figures 1C, 2C),
b-catenin in sporadic desmoid tumours 223
Copyright # 2001 John Wiley & Sons, Ltd. J Pathol 2001; 195: 222–228.
although MIB-1-LI ranged from 0 to 28.6 (mean: 3.0).
There was no correlation between MIB-1-LI and b-
catenin accumulation or cyclin D1 overexpression. On
the other hand, all cases demonstrated a broad
spectrum of immunoreactivity for PCNA (Figures 1D,
2D), ranging from 2.5 to 67.4 (mean: 31.8), and most
Figure 1. Immunohistochemical staining of b-catenin, cyclin D1, Ki-67 and PCNA in a desmoid tumour with b-catenin mutation atcodon 45. Most of the tumour cells throughout the lesion are positively stained for b-catenin (A: top left), and more than 50% of thetumour cell nuclei are positively stained for cyclin D1 (B: top right). Tumour cells positively stained for Ki-67 are scatteredthroughout the lesion (C: bottom left); in this case, the MIB-1-LI is 12.2. Most of the tumour cells throughout the lesion arepositively stained for PCNA (D: bottom right); in this case, the PCNA-LI is 67.4. Original magnifications, r57.5 (A–D)
Figure 2. Immunohistochemical staining of b-catenin, cyclin D1, Ki-67 and PCNA in a desmoid tumour without b-catenin mutation.Only a few tumour cells are positively stained for b-catenin (A: top left) and cyclin D1 (B: top right). Tumour cells positively stainedfor Ki-67 are almost undetectable (C: bottom left); in this case, the MIB-1-LI is 0.1. Only a few tumour cells are positively stained forPCNA (D: bottom right); in this case, the PCNA-LI is 2.5. Original magnifications, r57.5 (A–D)
224 T. Saito et al.
Copyright # 2001 John Wiley & Sons, Ltd. J Pathol 2001; 195: 222–228.
cases were stained homogeneously for PCNA. Eachgroup with widespread nuclear staining of b-catenin orcyclin D1 overexpression showed a higher PCNA-LIthan those without, and the differences were statisti-cally significant ( p=0.007, p=0.004, respectively:Tables 2 and 3).
Amplification of cyclin D1 gene
We were able to perform differential PCR successfullyin only 22 out of 38 cases, and we could detectamplification of the cyclin D1 gene in 13 of these(59.1%, Figure 3). There was no statistically significantassociation between tumour location and amplifica-tion of the cyclin D1 gene (p=0.39). Among 13 cases
with cyclin D1 gene amplification, nine showed over-expression of cyclin D1; eight of these also showedwidespread nuclear staining of b-catenin and theremaining one had b-catenin mutation. None of thefour cases with cyclin D1 gene amplification but with-out cyclin D1 overexpression showed widespreadnuclear staining of b-catenin. Moreover, among the eightcases with overexpression of cyclin D1 but withoutamplification of the cyclin D1 gene, six showed wide-spread nuclear staining of b-catenin, whereas the onecase without amplification of the cyclin D1 gene andwithout cyclin D1 overexpression did not show wide-spread nuclear staining of b-catenin (Table 4). How-ever, as a result, there was no statistically significantcorrelation between the amplification of the cyclin D1gene and cyclin D1 overexpression (p=0.360).
b-catenin gene mutation
We succeeded in obtaining PCR product in only 18 outof 38 cases. There were nine-point mutations in sevencases (38.9%, Figures 4 and 5), all of which weremissense mutations (Table 5), but no interstitial dele-tion was detected. There was no statistically significantdifference between tumour location and the incidenceof b-catenin mutation (p=0.39). All cases with b-catenin mutation showed overexpression of cyclin D1.
Table 1. Correlation between widespread nuclear stain-ing of b-catenin and cyclin D1 overexpression
Widespread nuclearstaining of b-catenin
(x) (+)
Cyclin D1 overexpression (x) 9 2 11
(+) 10 17 27
19 19 38
p=0.029
Table 2. Correlation between widespread nuclear stain-ing of b-catenin and PCNA-LI
PCNA-LI
Mean SD
Widespread nuclear staining of b-catenin (x) n=19 25.6 12.4
(+) n=19 37.9 14.1p=0.007
Table 3. Correlation between cyclin D1 overexpressionand PCNA-LI
PCNA-LI
Mean SD
Cyclin D1 overexpression (x) n=11 21.5 11.5(+) n=27 35.9 13.6
p=0.004
Figure 3. Differential PCR to detect cyclin D1 gene amplification in desmoid tumour. DNA product of cyclin D1 is upper 157bp,while DNA product of dopamine-D2-receptor is lower 128bp, respectively. Lane 1 shows differential PCR products in the controlHeLa cell line without amplification of the cyclin D1 gene. Lane 2 shows negative control. Cyclin D1 gene amplification is obvious inlanes 4, 5 and 6
Figure 4. Results of SSCP analysis of the b-catenin gene indesmoid tumours. Aberrantly migrating bands (arrows) can beobserved in lanes 2 (case 17), 3(case 19) and 7 (case 3)
Table 4. Correlation between amplification of cyclin D1gene and cyclin D1 overexpression
Amplification ofcyclin D1 gene
(x) (+)
Cyclin D1 overexpression (x) 1 4 5
(+) 8 9 17
9 13 22
p=0.360
b-catenin in sporadic desmoid tumours 225
Copyright # 2001 John Wiley & Sons, Ltd. J Pathol 2001; 195: 222–228.
In addition, all cases with b-catenin mutation showedamplification of the cyclin D1 gene, with the exceptionof one case which was not available for differentialPCR analysis. The distribution of b-catenin mutationin exon 3 observed in this study fell within a widerange, from codon 21 to codon 67.
Discussion
In this study, we have demonstrated for the first time a
statistically significant correlation between widespread
nuclear staining of b-catenin and cyclin D1 over-expression in desmoid tumours. However, widespread
nuclear staining of b-catenin or cyclin D1 over-expression did not affect proliferative activity as
assessed by MIB-1-LI. This finding may reflect the
essentially benign nature of desmoid tumors. On theother hand, PCNA has been known to be involved in
DNA repair when binding to cyclin D1 and p21, in
addition to DNA replication [22–24]. PCNA wasdiffusely expressed in sporadic desmoid tumours, and
interestingly, each group with widespread nuclearstaining of b-catenin or cyclin D1 overexpression
showed higher PCNA-LI than those without. These
findings may suggest that PCNA has an important rolein DNA repair and the inhibition of DNA replication
in sporadic desmoid tumours; PCNA may therefore be
expressed more strongly in cases with overexpressionof b-catenin and cyclin D1. Furthermore, the cases
with a higher MIB-1-LI and a higher local recurrencerate may have some abnormalities in the function of
PCNA.There were 10 cases with cyclin D1 overexpression
which did not demonstrate widespread nuclear stainingof b-catenin. Although we could only perform muta-
tional analysis for three of these ten cases, two hadb-catenin mutations; some of the remaining seven cases
may also have had b-catenin mutations, bearing in
mind that desmoid tumours have a rather highfrequency of such events. In addition, differential
PCR analysis was possible for five out of nine cases
without widespread nuclear staining of b-catenin andwithout cyclin D1 overexpression; four of these cases
showed amplification of the cyclin D1 gene. Further-more, all six cases with b-catenin mutation showed
overexpression of cyclin D1, together with amplifica-
tion of the cyclin D1 gene. One additional case was notavailable for differential PCR analysis. These results
strongly suggest that the cyclin D1 gene is frequently
amplified in desmoid tumours, but this alone cannotaccount for the immunohistochemically detectable
expression of cyclin D1. This further suggests that
a
b
Figure 5. Sequencing results for exon 3 of b-catenin in desmoidtumours. (A) Tumour sequences showing the substitution ATTfor ATC (arrow, left) at codon 35 and the substitution ATA forACA (arrow, centre) at codon 42 in case 17. Tumour sequenceshowing the substitution TTT for TCT (arrow, right) at codon45 in case 3. (B) Tumour sequences showing the substitutionGAA for GAG (arrow, left) at codon 65 and the substitutionAAA for GAA (arrow, right) at codon 67 in case 19
Table 5. Correlation of cyclin D1 overexpression and b-catenin mutation in sporadic desmoid tumours
Case
Age(years)/
sex Location Codon
Nucleotide
change
Amino acid
change
b-catenin
aberrant staining
Amplification
of cyclin D1 gene
Cyclin D1
overexpression
3 34/M Left thigh 45 TCT to TTT Ser to Phe >75% (+) (+)
17 31/F Abdominal wall 35 ATC to ATT Ile to Ile <75% (+) (+)
42 ACA to ATA Thr to Ile19 25/F Right upper arm 65 GAG to GAA Glu to Glu >75% (+) (+)
67 GAA to AAA Glu to Lys
24 12/F Chest wall 53 GAG to AAG Glu to Lys >75% (+) (+)32 68/M Right lower leg 21 GCT to ACT Ala to Thr <75% ND (+)
33 49/F Back 21 GCT to ACT Ala to Thr >75% (+) (+)
34 73/M Back 45 TCT to TTT Ser to Phe >75% (+) (+)
N D: No data.
226 T. Saito et al.
Copyright # 2001 John Wiley & Sons, Ltd. J Pathol 2001; 195: 222–228.
cyclin D1 expression is activated to some extent byb-catenin accumulation in sporadic desmoid tumours.The effect of this pathway is reinforced when combinedwith amplification of the cyclin D1 gene.
The reported values of the incidence of b-cateninmutation in sporadic desmoid tumours without FAPare quite similar [5,9]. In this study, we found b-cateninmutation in 7 out of 18 cases (38.9%), lower, thoughnot significantly, than previously reported values, [5,9]but the most striking difference concerned the muta-tional sites, which had a wider range than in previousstudies [5,8,9]. Previously, reports were confined tocodon 41 or codon 45 [5,8,9], whereas only two out ofour seven cases had a b-catenin mutation at codon 45,while the other five involved codons 21, 35, 42, 53, 65and 67. Furthermore, most of the previously reportedb-catenin point mutation sites in other human benignand malignant tumours were at codons 32 (Asp), 33(Ser), 34 (Gly), 37 (Ser), 41 (Thr) and 45 (Ser) [25–31].To our knowledge, only anaplastic thyroid carcinomascontain such a wide range of b-catenin mutations,falling between codon 17 (Asp) and codon 60 (Ser)[32], although mutations at codons 21, 35, 53, 65 and67 had not previously been reported. All of our caseswith b-catenin mutation showed nuclear expression ofb-catenin and overexpression of cyclin D1, althoughtwo did not show widespread nuclear staining ofb-catenin. However, the function of b-catenin mutationsother than those affecting serine/threonine residues ortheir neighbouring sites is still unclear, since thepossibility that APC gene mutations were involved inthese cases could not be completely ruled out, despitethe lower frequency of such mutations in sporadicdesmoid tumours [7].
In conclusion, the most striking finding in this studywas that in sporadic desmoid tumours, b-cateninnuclear expression correlated with cyclin D1 over-expression, but not with cell proliferation as assessedby MIB-1-LI. This could be an in vivo model systemfor the APC-b-catenin-Tcf pathway. In addition, b-catenin mutations in desmoid tumours occurred at anunusually wide range of sites within the gene.
Acknowledgements
This work was supported in part by a Grant-in-Aid for Cancer
Research from the Fukuoka Cancer Society and Grants-in-Aid
for General Scientific Research from the Ministry of Education,
Science, Sports and Culture (09470052, 12670167) of Japan. We
are grateful to Miss M. Okano for her excellent technical
assistance. We thank Miss Katherine Miller (Royal English
Language Centre, Fukuoka, Japan) for revising the English used
in this article.
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