Expression of Bcl-2 family proteins and associated clinicopathologic factors predict survival...

Expression of Bcl-2 family proteins and association withclinicopathological characteristics of oral squamous cellcarcinoma

Claudia Malheiros Coutinho-Camillo,1 Silvia Vanessa Lourenco,2 Ines Nobuko Nishimoto,3

Luiz Paulo Kowalski3 & Fernando Augusto Soares1,2

1Department of Pathology, Hospital A.C. Camargo, 2Department of General Pathology, Dental School, University of Sao

Paulo, and 3Department of Otorhinolaryngology and Head and Neck Surgery, Hospital A.C. Camargo, Sao Paulo, SP, Brazil

Date of submission 11 May 2009Accepted for publication 19 November 2009

Coutinho-Camillo C M, Lourenco S V, Nishimoto I N, Kowalski L P & Soares F A

(2010) Histopathology 57, 304–316

Expression of Bcl-2 family proteins and association with clinicopathological characteristics oforal squamous cell carcinoma

Aims: To characterize the expression of proteins thatinhibit (Bcl-2, Bcl-x, Bcl-xL, Bcl-2-related protein A1,BAG-1) or promote (Bak, Bax, Bim ⁄ Bod, Bim-Long,Bad, Bid, PUMA) apoptosis and determine possiblecorrelations between the expression of these proteinsand clinicopathological features of oral squamous cellcarcinoma (OSCC).Methods and results: Two-hundred and twenty-ninecases of OSCC, arranged in a tissue microarray, wereimmunohistochemically analysed. The results demon-strated that the absence of vascular invasion wasassociated with increased expression of Bak, Bax,Bcl-xL, Bcl-2-related protein and PUMA. Increasedexpression of Bim ⁄ Bod and BAG-1 was associated

with the presence of perineural infiltration. Anincrease in Bid and Bim-Long expression was associ-ated with moderately to well-differentiated tumours.Increased expression of the Bcl-2-related protein andPUMA was associated with tumours occurring in thefloor of mouth and increased expression of PUMAwas also associated with recurrence of the tumour.Multivariate Cox analysis demonstrated that PUMAand Bim-Long were independent factors in prognosisof OSCC.Conclusions: Our results showed the involvement of theBcl-2 family of proteins in OSCC tumorigenesis andsuggest that the expression of apoptotic moleculesmight be used as a prognostic indicator for OSCC.

Keywords: apoptosis, Bcl-2, immunohistochemistry, oral cancer, tissue microarray

Abbreviations: cIAP, cellular inhibitor of apoptosis protein; DFS, disease-free survival; HNSCC, head and necksquamous cell carcinoma; OS, overall survival; OSCC, oral squamous cell carcinoma; PI3K, phosphoinositide3-kinase; SCC, squamous cell carcinoma; TMA, tissue microarray

Introduction

Squamous cell carcinoma (SCC) constitutes at least90% of all oral malignancies.1–3 According to theWorld Health Organization,4 oral squamous cell carci-noma (OSCC) is the eighth most common cancer

worldwide, with geographical variations. It is highlyprevalent in Brazil, where it had an estimated occur-rence of 14 120 new cases of oral cancer in 2009.5

OSCC is characterized by significant morbidity andmortality rates, and in spite of the vast amount ofresearch and advances in the field of oncology andsurgery, mortality rates remain virtually unchanged.This lack of a decrease in mortality rates drives thesearch for factors of prognostic relevance that wouldallow for OSCC treatment to be to tailored to themanagement of individual patients.1,6,7

Address for correspondence: C M Coutinho-Camillo, Hospital AC

Camargo, Departamento de Anatomia Patologica, Rua Professor

Antonio Prudente, 109 1 andar, Liberdade, Sao Paulo,

CEP: 01509-010, SP, Brazil. e-mail: [email protected]

� 2010 Blackwell Publishing Limited.

Histopathology 2010, 57, 304–316. DOI: 10.1111/j.1365-2559.2010.03621.x

Apoptosis is a genetically programmed type of celldeath, which primarily functions to eliminate senescentor altered cells that are useless or harmful for amulticellular organism. In contrast, perturbations inapoptotic mechanisms that promote excessive or defi-cient programmed cell death have been linked to a widearray of pathological conditions.8 Mounting evidencesuggests that oral carcinogenesis is correlated with aprogressive accumulation of genetic alterations inmolecules that play crucial roles during apoptosis.9–13

The Bcl-2 protein family consists of two opposinggroups of proteins: death antagonists and death agon-ists, each differing in their tissue- and activation-dependent expression patterns as well as in theirstructure. The activity of the Bcl-2 protein family ispartly regulated by the formation of homo- andheterodimers between family members.14,15 One ofthe most studied anti-apoptotic proteins is Bcl-2. Itsabnormal expression has been reported in a number ofsolid tumours such as lung, thyroid, breast, ovary,stomach and colonic carcinomas.16

Most studies of the Bcl-2 family members expressedin oral tissues have predominantly concentrated onBcl-2 and Bax and their results are controversial.10,16–20

Using a tissue microarray (TMA) constructed with 229cases of OSCC, the aim of the present study was tocharacterize the expression of proteins that inhibit(Bcl-2, Bcl-x, Bcl-xL, Bcl-2-related protein A1, BAG-1)or promote (Bak, Bax, Bim ⁄ Bod, Bim-Long, Bad, Bidand PUMA) apoptosis and determine possible correla-tions between the expression of these proteins andclinicopathological features of OSSC.

Material and methods

tissue samples

Paraffin-embedded tissue samples from 229 OSCC caseswere obtained from the files of the Department ofPathology of the Hospital A. C. Camargo (Sao Paulo,Brazil). All retrieved cases had been under treatment atthe hospital for at least 5 years. Clinical and histolog-ical details of the cases are provided in Table 1.Informed consent was obtained from all patients andthe Institutional Ethics Committee approved this study(Protocol no. 985 ⁄ 07).

tissue microarray

For TMA construction, haematoxylin and eosin sectionswere analysed and a representative area of the tumourwas marked on the slide. The tissues corresponding toselected areas were sampled from the donor block, using

a Tissue Microarrayer (Beecher Instruments, SilverSprings, MD, USA). The cores were taken from the deeparea of the carcinomas. Each sample was arrayed oncewith a 1.0 mm diameter core spaced 0.2 mm apart, andtwo TMAs with different core regions were constructed.After the array was completed, TMA blocks weresectioned at a thickness of 4 lm.

Table 1. Summary of the clinicopathological characteristicsof the head and neck cancer patients

Characteristic CategoryNumberof patients

Age (years) £60 151

>60 78

Gender Male 194

Female 35

Race White 199

Non-White 30

Tobacco smoking No 19

Yes 182

Alcohol consumption No 45

Yes 153

Clinical stage I ⁄ II 77

III ⁄ IV 152

Tumour site Oral tongue 122

Floor of mouth 55

Other 52

Lymph nodemetastasis (pN)

No 92

Yes 112

Perineural infiltration No 129

Yes 86

Vascular invasion No 72

Yes 141

Histological grade Poorly differentiated 177

Moderately ⁄well-differentiated

46

Recurrence No 123

Yes 106

Bcl-2 family proteins in OSCC 305

� 2010 Blackwell Publishing Ltd, Histopathology, 57, 304–316.

immunohistochemistry

The expression of Bcl-2, Bcl-x, Bcl-xL, Bcl-2-relatedprotein A1, BAG-1, Bak, Bax, Bim ⁄ Bod, Bim-Long,Bad, Bid and PUMA was investigated in OSCC tissuesamples arranged in a TMA. Immunohistochemistrywas performed on duplicate tissue slides from twodifferent cores; duplicate sections were 40 lm apart.The slides were deparaffinized, re-hydrated and thensubjected to antigen retrieval. Details on antigenretrieval methods as well as primary antibodies clones,source and titre are given in Table 2. The sectionswere then incubated in 3% aqueous hydrogen peroxidefor 15 min to quench endogenous peroxidase activityand with Protein Block Serum-Free (Dako, Carpinteria,CA, USA) for 20 min at room temperature to suppressnon-specific binding of subsequent reagents. Thereaction was followed with incubation of the primaryantibody with the tissue sections for 2 h at roomtemperature. The antigen–antibody complexes werevisualized using Advance detection system (Dako)followed by incubation with 3¢3 diaminobenzidinetetrachloride (Dako) for 5 min. The sections were thencounterstained with Mayer’s haematoxylin, dehy-drated and mounted with a glass coverslip andxylene-based mounting media. The results were quan-titatively analysed using an automated imaging system(ACIS III; Dako), which detects levels of hue, satura-tion and luminosity, converting this signal into anumerical density measurement (intensity of immu-

noreactivity) that ranges from 0 to 256 (Table 3). Thefollowing scores were assigned: ‘low expression’ forexpression levels up to the median value of intensity of

Table 2. Primary serum, clones, source, working titre and antigen retrieval

Primary serum Clone Source Working titre Antigen retrieval

Bcl-2 124 Dako (Carpinteria, CA, USA) 1:200 Citrate pH 6.0

Bcl-x Polyclonal Dako 1:500 Citrate pH 6.0

Bcl-xL 7D9 Chemicon (Billerica, MA, USA) 1:2000 Citrate pH 6.0

Bcl-2-related protein A1 EP517Y Epitomics (Burlingame, CA, USA) 1:150 Citrate pH 6.0

BAG-1 3.10G3E2 Neomarkers (Fremont, CA, USA) 1:100 EDTA ⁄ Tris pH 9.0

Bak Polyclonal Dako 1:300 Citrate pH 6.0

Bax Polyclonal Dako 1:400 Citrate pH 6.0

Bim ⁄ Bod Polyclonal Neomarkers 1:150 EDTA ⁄ Tris pH 9.0

Bim-Long 5E5 Chemicon 1:100 Citrate pH 6.0

Bad Y208 Epitomics 1:1500 EDTA ⁄ Tris pH 9.0

Bid BH3 Epitomics 1:200 Citrate pH 6.0

PUMA EP512Y Epitomics 1:250 Citrate pH 6.0

Table 3. Threshold settings for immunohistochemical analy-sis on ACIS III system

Marker Hue Luminosity Saturation

Bcl-2 185 ⁄ 171* 216 ⁄ 146 37 ⁄ 3

Bcl-x 171 ⁄ 18 200 ⁄ 175 21 ⁄ 6

Bcl-xL 179 ⁄ 26 189 ⁄ 156 21 ⁄ 4

Bcl-2-relatedprotein A1

234 ⁄ 160 196 ⁄ 164 25 ⁄ 4

BAG-1 162 ⁄ 64 192 ⁄ 178 13 ⁄ 2

Bak 128 ⁄ 34 210 ⁄ 165 19 ⁄ 2

Bax 189 ⁄ 167 149 ⁄ 55 68 ⁄ 20

Bim ⁄ Bod 241 ⁄ 36 206 ⁄ 189 11 ⁄ 5

Bim-Long 163 ⁄ 40 196 ⁄ 178 30 ⁄ 3

Bad 158 ⁄ 11 183 ⁄ 163 24 ⁄ 4

Bid 188 ⁄ 11 216 ⁄ 204 10 ⁄ 3

PUMA 171 ⁄ 0 201 ⁄ 180 31 ⁄ 0

*Upper and lower settings, respectively.

306 C M Coutinho-Camillo et al.


reactivity, and ‘high expression’ for expression levelsabove the median value of intensity of reactivity.Negative controls were performed by incubation of thetissue sections with non-immune serum. Positivecontrols were employed according to the manufac-turer’s recommendations. For establishment of pat-terns of expression, the following scores were assigned:anti-apoptotic pattern, for cases with high expressionof three anti-apoptotic proteins and low expression ofat least three pro-apoptotic proteins; and pro-apoptoticpattern, for cases with high expression of four pro-apoptotic proteins and low expression of at least threeanti-apoptotic proteins.

statist ical analysis

Analysis of the association between protein levels andthe demographic and clinicopathological characteris-tics of the patients was performed using the Mann–Whitney U-test or one-way anova test using theBonferroni correction, considering protein expressionas a continuous variable. We analysed the differencesin protein expression among the following differentgroups: clinical stage (I ⁄ II and III ⁄ IV), tumour site(oral tongue, floor of mouth or other sites), lymph nodemetastasis (yes or no), vascular invasion (yes or no),perineural infiltration (yes or no), histological grade(poorly differentiated and moderately ⁄ well differenti-ated) and recurrence. Overall (OS) and disease-freesurvival (DFS) probabilities were calculated based onthe Kaplan–Meier method with log rank score fordetermining statistical significance. Relative risk wasevaluated by the multivariate Cox proportional hazardsmodel. The significance level was 5% for all statisticaltests. Statistical analyses were performed using Graph-

Pad Prism version 5.02 for Windows (GraphPadSoftware, San Diego CA, USA; http://www.graphpad.com).

Results

All anti-apoptotic (Bcl-2, Bcl-x, Bcl-xL, Bcl-2-relatedprotein A1, BAG-1) and pro-apoptotic (Bak, Bax,Bim ⁄ Bod, Bim-Long, Bad, Bid, PUMA) proteins testedwere present in the OSCC samples studied. Theseproteins displayed variable expression levels and all ofthem could be seen by cytoplasmic immunoreactivityin neoplastic cells and in the controls (Figure 1). Allproteins were also present in normal oral squamousmucosa in the mid and lower layers. The majority ofthe proteins displayed median expression values innormal cells lower than that found in neoplastic cells,

with the exception of Bcl-2, Bcl-x and PUMA. Theseresults are described in Table 4.

anti-apoptotic proteins

Our results demonstrated that increased expression ofthe anti-apoptotic proteins Bcl-xL and Bcl-2-relatedprotein was associated with the absence of vascularinvasion (P = 0.0003, P = 0.0010, respectively). In-creased expression of BAG-1 was associated with thepresence of perineural infiltration (P = 0.0051) andincreased expression of Bcl-2-related protein was asso-ciated with tumour occurring in the floor of mouth(P = 0.0152, when compared with other sites and notwith oral tongue). Increased expression of Bcl-2-relatedprotein was also associated with early-stage tumours(stages I and II) (P = 0.0509). These statistical resultsare shown in Table 5.

The OS rate was significantly different betweenpatients with low and high expression of Bcl-2-relatedprotein (P = 0.0262). Median survival in 5 years was33.7% for patients with low expression and 48.3% forpatients with high expression of Bcl-2-related protein.DFS rate was significantly different between patientswith low and high expression of Bcl-x (P = 0.0376).Median survival in 5 years was 56.7% for patients withlow expression and 40.1% for patients with highexpression of Bcl-x.

Using the Cox regression model, we performedmultivariate analysis to assess the independent predic-tive value of Bcl-2-related protein for OS and Bcl-x forDFS. None of the variables could independently be aprognostic factor for OSCC patients.

pro-apoptotic proteins

Our results demonstrated that increased expression ofthe pro-apoptotic proteins Bak, Bax and PUMA wasassociated with the absence of vascular invasion(P £ 0.0001, P = 0.0192, P = 0.0523, respectively).Increased expression of Bim ⁄ Bod was associated withthe presence of perineural infiltration (P = 0.0028).Increased expression of Bid and Bim-Long was associatedwith moderately ⁄ well-differentiated tumours (P =0.0519, P = 0.0143, respectively). Additionally, in-creased expression of Bax was associated with tumouroccurring in the oral tongue (P = 0.0052, when com-pared with other sites and not with floor of mouth).Increased expression of PUMA was associated withtumour occurring in the floor of the mouth (P = 0.0523,when compared with the oral tongue and not with othersites). These statistical results are shown in Table 5.



Increased expression of PUMA was associated withrecurrence of the tumour (P = 0.0513), and the DFSrate was significantly different between patients withlow and high expression of PUMA (P = 0.0291).Median survival in 5 years was 54.7% for patients

with low expression and 41.9% for patients with highexpression of PUMA.

The OS rate was significantly different betweenpatients with low and high expression of Bak(P = 0.0085). Median survival in 5 years was 34.5%

A B C

D E F

G H I

J K L

Figure 1. Expression of the Bcl-2 family of proteins in oral squamous cell carcinoma. A, Bcl-2. B, Bcl-x. C, Bcl-xL. D, Bcl-2-related protein.

E, BAG-1; F, Bak. G, Bax. H, Bim ⁄ Bod. I, Bim-Long. J, Bad. K, Bid. L, PUMA.



for patients with low expression and 48.8% for patientswith high expression of Bak.

Using the Cox regression model, we performedmultivariate analysis to assess the independent predic-tive value of variables exhibiting significance or nearsignificance for OS [Bak (P = 0.0085) and Bim-Long(P = 0.0935)] and for DFS [PUMA (P = 0.0291) andBak (P = 0.0726)]. Expression of PUMA and Bim-Longcould be independent prognostic factors for OSCCpatients. These results are described in Tables 6 and 7.

Considering low and high expression of the differentproteins, we further grouped the cases with an overallpattern of expression in favour of or against apoptosis.However, OS rate was not significantly differentbetween cases with an anti- or pro-apoptotic patternof expression (P = 0.4307).

Discussion

Alterations in the expression level of apoptosis-associ-ated proteins have been reported in several cancers,including oral cancer.9–13 In this study, using TMAanalysis of 229 cases, we have demonstrated that theexpression of apoptotic proteins varies among the casesof OSCC analysed.

TMAs are a powerful research tool capable ofsimultaneously processing large numbers of tumour

specimens to detect expression levels of multiple pro-teins using immunohistochemical analysis as is com-monly accepted in clinical practice. This methodologyalso gives experimental uniformity, and analysing alarge number of samples improves statistical precisionin addition to enhanced speed and quality of analysis.The most obvious drawback of this technology wasinitially believed to be the possibility that smallcylinders might not adequately represent whole sec-tions because of intratumour heterogeneity of proteinexpression. However, increasing the number of corescollected from the sample and ⁄ or increasing the corediameter can resolve this limitation.21 Furthermore,the use of an automated scoring apparatus enables amore objective, quantitative and observer-independentmeans of acquiring staining result measurements dueto its higher accuracy, sensitivity and better reproduc-ibility of data.22

The Bcl-2 family of proteins consists of two catego-ries of proteins: death antagonists (Bcl-2, Bcl-x, Bcl-xL,Bcl-2-related protein A1, BAG-1) and death agonists(Bak, Bax, Bim ⁄ Bod, Bim-Long, Bad, Bid, PUMA).Death agonists includes two types of molecules: BH3-only proteins (Bim ⁄ Bod, Bim-Long, Bad, Bid, PUMA)that are sensors of cellular damage and can engage andinactivate death antagonists, allowing the second typeof death agonist molecules (Bak, Bax) to be activated.

Table 4. Results of immunostaining quantitatively analysed using an automated imaging system (ACIS III)

Protein

Neoplastic cells Normal cells

Intensity (range)Median valueof intensity Intensity (range)

Median valueof intensity

Bcl-2 52.0–80.0 55.0 56.0–57.0 57.0

Bcl-x 66.0–111.0 79.2 83.3–96.0 84.0

Bcl-xL 96.0–155.5 128.7 80.7–95.0 83.0

Bcl-2-related protein A1 73.0–128.0 91.0 74.7–87.0 79.2

BAG-1 ⁄ RAP46 69.0–102.0 78.0 68.0–76.0 74.6

Bak 91.5–142.0 112.0 88.5–101.3 98.5

Bax 78.0–135.0 107.0 74.0–75.0 74.7

Bim ⁄ Bod 65.0–98.3 77.2 65.3–71.5 66.5

BIM-Long 81.0–125.0 99.5 81.0–85.5 82.9

Bad (N-term) 70.0–118.5 83.5 75.0–84.5 78.5

Bid 63.0–96.5 74.3 62.3–65.0 63.5

Puma 61.0–116.5 82.8 97.5–117.0 113.8



Tab

le5.

Ass

oci

atio

nbet

wee

npro

tein

expre

ssio

nan

dcl

inic

opat

holo

gic

alch

arac

terist

ics

of

the

hea

dan

dnec

kca

nce

rpat

ients

Char

acte

rist

icC

ateg

ory

Bcl

-2Bcl

-xBcl

-xL

Bcl

-2re

late

dpro

tein

A1

BA

G-1

Clin

ical

stag

eI

⁄II

55.0

(n=

53)*

79.5

(n=

56)

129.2

(n=

60)

94.0

(n=

59)

78.0

(n=

55)

III

⁄IV

55.0

(n=

131)

79.0

(n=

135)

128.2

(n=

136)

90.3

(n=

137)

78.0

(n=

132)

P=

0.3

926

P=

0.4

299

P=

0.4

069

P=

0.0

509

P=

0.5

610

Tum

our

site

Ora

lto

ngue

55.0

(n=

92)

79.0

(n=

99)

126.7

(n=

103)

91.0

(n=

99)

78.0

(n=

95)

Floor

of

mouth

55.3

(n=

44)

81.7

(n=

47)

132.8

(n=

46)

91.9

(n=

50)

78.5

(n=

45)

Oth

er55.3

(n=

48)

79.2

(n=

45)

127.0

(n=

47)

89.3

(n=

47)

77.5

(n=

47)

P=

0.0

550

P=

0.1

884

P=

0.3

296

P=

0.0

152

P=

0.5

754

Lym

ph

node

met

asta

sis

No

55.0

(n=

75)

79.2

(n=

74)

132.0

(n=

77)

92.0

(n=

78)

78.0

(n=

74)

Yes

55.0

(n=

96)

79.1

(n=

100)

127.0

(n=

100)

90.0

(n=

100)

78.3

(n=

96)

P=

0.7

122

P=

0.9

781

P=

0.1

625

P=

0.1

055

P=

0.6

621

Per

ineu

ral

infiltra

tion

No

55.0

(n=

104)

79.2

(n=

106)

128.4

(n=

108)

91.5

(n=

109)

77.4

(n=

102)

Yes

55.3

(n=

72)

79.4

(n=

74)

129.1

(n=

76)

91.2

(n=

76)

79.6

(n=

74)

P=

0.4

326

P=

0.8

948

P=

0.6

069

P=

0.9

833

P=

0.0

051

Vas

cula

rin

vasi

on

No

55.3

(n=

59)

79.2

(n=

58)

134

(n=

61)

94.5

(n=

60)

78.2

(n=

58)

Yes

55.0

(n=

115)

79.3

(n=

120)

124.7

(n=

122)

90.0

(n=

124)

78.0

(n=

117)

P=

0.1

175

P=

0.9

307

P=

0.0

003

P=

0.0

010

P=

0.3

087

His

tolo

gic

algra

de

Poorly

diffe

rentiat

ed55.0

(n=

141)

79.3

(n=

147)

128.7

(n=

155)

91.0

(n=

152)

78.0

(n=

144)

Moder

atel

y⁄

wel

l-diffe

rentiat

ed55.5

(n=

37)

78.4

(n=

38)

129.3

(n=

35)

91.3

(n=

38)

79.0

(n=

37)

P=

0.2

705

P=

0.2

224

P=

0.5

091

P=

0.4

874

P=

0.2

535

Rec

urr

ence

No

55.3

(n=

99)

78.6

(n=

102)

130.3

(n=

104)

91.3

(n=

103)

78.0

(n=

98)

Yes

55.0

(n=

85)

79.8

(n=

89)

127.3

(n=

92)

91.0

(n=

93)

78.0

(n=

89)

P=

0.3

997

P=

0.2

325

P=

0.6

670

P=

0.3

088

P=

0.9

946



Tab

le5.

(Conti

nued)

Char

acte

rist

icC

ateg

ory

Bak

Bax

Bim

⁄Bod

Bim

-Long

Bad

Bid

PU

MA

Clin

ical

stag

eI

⁄II

112.4

(n=

58)*

108.6

(n=

68)

77.2

(n=

54)

99.5

(n=

61)

83.5

(n=

51)

73.7

(n=

56)

83.0

(n=

59)

III

⁄IV

111.6

(n=

130)

106.5

(n=

140)

77.2

(n=

135)

99.4

(n=

138)

83.8

(n=

128)

74.5

(n=

133)

82.5

(n=

137)

P=

0.5

974

P=

0.3

733

P=

0.8

876

P=

0.9

979

P=

0.5

586

P=

0.2

991

P=

0.8

952

Tum

our

site

Ora

lto

ngue

113.0

(n=

98)

109.0

(n=

107)

77.0

(n=

96)

101.0

(n=

103)

83.9

(n=

92)

74.4

(n=

96)

81.0

(n=

102)

Floor

of

mouth

112.5

(n=

45)

107.5

(n=

51)

78.5

(n=

45)

98.5

(n=

47)

84.7

(n=

41)

73.3

(n=

46)

85.4

(n=

47)

Oth

er110.8

(n=

45)

102.7

(n=

50)

76.9

(n=

45)

98.0

(n=

49)

83.1

(n=

46)

75.3

(n=

47)

83.7

(n=

47)

P=

0.0

709

P=

0.0

052

P=

0.2

701

P=

0.4

277

P=

0.8

968

P=

0.9

803

P=

0.0

523

Lym

ph

node

met

asta

sis

No

113.0

(n=

73)

107.0

(n=

83)

77.0

(n=

75)

99.0

(n=

79)

84.2

(n=

72)

74.8

(n=

72)

82.1

(n=

78)

Yes

111.5

(n=

98)

106.3

(n=

103)

77.5

(n=

100)

99.5

(n=

101)

83.3

(n=

95)

74.6

(n=

102)

83.0

(n=

100)

P=

0.7

584

P=

0.4

545

P=

0.2

323

P=

0.3

792

P=

0.6

886

P=

1.0

000

P=

0.5

076

Per

ineu

ral

infiltra

tion

No

112.0

(n=

104)

106.8

(n=

116)

76.5

(n=

104)

98.2

(n=

113)

83.1

(n=

98)

74.3

(n=

103)

82.8

(n=

110)

Yes

113.1

(n=

74)

107.8

(n=

79)

78.5

(n=

73)

101.0

(n=

74)

85.0

(n=

69)

74.5

(n=

76)

82.2

(n=

74)

P=

0.3

502

P=

0.1

289

P=

0.0

028

P=

0.0

943

P=

0.3

104

P=

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471

P=

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644

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116.0

(n=

59)

112.5

(n=

65)

77.5

(n=

55)

102.0

(n=

65)

86.0

(n=

51)

74.5

(n=

57)

83.7

(n=

60)

Yes

109.7

(n=

119)

105.4

(n=

128)

77.1

(n=

120)

98.3

(n=

120)

83.3

(n=

114)

74.0

(n=

121)

81.9

(n=

122)

P<

0.0

001

P=

0.0

192

P=

0.1

912

P=

0.1

143

P=

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568

P=

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749

P=

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523

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(n=

146)

106.7

(n=

161)

77.0

(n=

146)

98.1

(n=

154)

83.5

(n=

139)

73.7

(n=

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82.2

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107.3

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78.0

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105.0

(n=

39)

85.9

(n=

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77.0

(n=

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38

(n=

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)

P=

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240

P=

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998

P=

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346

P=

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143

P=

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177

P=

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519

P=

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Rec

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ence

No

112.8

(n=

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107.0

(n=

111)

77.3

(n=

98)

98.2

(n=

105)

84.5

(n=

94)

74.4

(n=

98)

81.8

(n=

104)

Yes

111.5

(n=

89)

107.0

(n=

97)

77.0

(n=

91)

99.8

(n=

94)

82.3

(n=

85)

74.3

(n=

91)

84.5

(n=

92)

P=

0.7

514

P=

0.9

466

P=

0.2

367

P=

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909

P=

0.4

269

P=

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932

P=

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513

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Most studies examining the Bcl-2 family in oraltissues have predominantly concentrated on Bcl-2 andBax. Some have shown an increase in expression ofBcl-2 in dysplastic lesions and in OSCC,9,17,20,23–25

whereas others have detected sporadic10,16,19 or lack ofexpression.26 Other studies have indicated that Bcl-2was involved in tumour growth and angiogenesis andcould be an indicator of poor prognosis.27,28 In thisstudy, we found that expression of Bcl-2 in OSCC was

not significantly associated with any of the clinico-pathological parameters analysed.

BAG-1 is a multifunctional protein that blocksapoptosis and interacts with several types of proteins,including Bcl-2 family proteins.29 Our results demon-strate an increase in BAG-1 expression associated withthe presence of perineural infiltration in OSCC. Somestudies have suggested that analysis of BAG-1 expres-sion may be useful in establishing a prognosis for

Table 6. Five-year global survival probabilities of the head and neck cancer patients and relative risk with estimated confidenceinterval of 95% by Cox regression models

Variables CategoriesFive-yearprobability (%) P-value

HR

Crude (95% CI) Multivariate (95% CI)

Gender Male 34.5 0.0021 1.0 (ref) 1.0 (ref)

Female 62.9 0.42 (0.2, 0.7) 0.41 (0.2, 0.8)

Lymph nodes No 58.2 0.0327 1.0 (ref) 1.0 (ref)

Yes 29.4 2.30 (1.6, 3.3) 2.00 (1.3, 3.0)

Positive lymph nodes No 46.8 <0.0001 1.0 (ref) 1.0 (ref)

Yes 13.6 2.22 (1.4, 3.6) 2.68 (1.4, 5.0)

Vascular invasion No 61.2 0.0005 1.0 (ref) 1.0 (ref)

Yes 35.7 1.94 (1.3, 2.8) 1.83 (1.1, 3.0)

Bim-Long Low expression (£99.5) 43.7 0.0935 1.0 (ref) 1.0 (ref)

High expression (>99.5) 38.6 1.34 (1.0, 1.9) 1.69 (1.1, 2.5)

P-value obtained by log rank test.

HR, hazard ratio.

Table 7. Five-year disease-free survival probabilities of the head and neck cancer patients and relative risk with estimatedconfidence interval of 95% by Cox regression models

Variables Categories5-year DFSprobability (%) P-value

HR

Crude (95%CI) Multivariate (95%CI)

Clinical stage I ⁄ II 58.3 0.0327 1.0 (ref) 1.0 (ref)

III ⁄ IV 43.5 1.59 (1.0, 2.4) 1.99 (1.2, 3.2)

Positive lymph nodes No 54.2 <0.0001 1.0 (ref) 1.0 (ref)

Yes 0.0 5.53 (3.4, 9.0) 6.43 (3.7, 11.1)

PUMA Low expression (£82.8) 54.7 0.0291 1.0 (ref) 1.0 (ref)

High expression (>82.8) 41.9 1.58 (1.0, 2.4) 1.64 (1.1, 2.5)

P-value obtained by log rank test.

HR, hazard ratio; DFS, disease-free survival.



patients with oral SCCs and in predicting the metastaticpotential of SSCs.30–33

The protein Bcl-x has two isoforms, Bcl-xL andBcl-xS, the former repressing cell death and the latterfavouring cell death. We evaluated Bcl-x expression byusing two different antibodies: one that does notdistinguish between the short and long isoforms andan antibody against Bcl-xL. Increased expression of Bcl-xL was associated with the absence of vascularinvasion. Although expression of Bcl-x was not signif-icantly associated with any of the clinicopathologicalparameters analysed, DFS rate was significantly betterin patients with low expression of Bcl-x. However, onmultivariate analysis, expression of Bcl-x did not proveto be an independent prognostic factor for OSCCpatients. In contrast, Camisasca et al.20 found thatBcl-x expression was indicative of longer DFS, suggest-ing that Bcl-2 and Bcl-x proteins may be under theregulation of post-translational mechanisms.

Bcl-2-related protein A1 (Bfl-1) is an anti-apoptoticBcl-2 family member that has been shown to reducethe release of cytochrome c from the mitochondria,block caspase activation and suppress apoptosisinduced by p53.34 The current study is thus far theonly study regarding Bfl-1 expression and OSCC. Parket al.35 analysed the expression of the Bfl-1 gene byNorthern blot and found that it was highly expressed ingastric, colonic and stomach cancer but mildlyexpressed in breast cancer, bone and soft tissuesarcoma and ovarian cancer. In our study, increasedexpression of Bcl-2-related protein A1 was associatedwith tumours occurring in the floor of the mouth.Although Bcl-2 A1 is an anti-apoptotic protein, its highexpression was also associated with the absence ofvascular invasion and with early-stage (I ⁄ II) tumours.Furthermore, the OS rate was significantly better inpatients with high expression of Bcl-2 A1. However, onmultivariate analysis, expression of Bcl-2 A1 did notprove to be an independent prognostic factor for OSCCpatients. The activity of the Bcl-2 family of proteins canbe diversely regulated by post-translational modifica-tions, such as phosphorylation or proteolytic cleavage.Some reports have demonstrated that various proteasescleave several Bcl-2 family proteins to produce largeC-terminal fragments with potent pro-apoptotic activ-ity. Ko et al.36 showed that Bfl-1 was converted into apro-apoptotic protein by using a green fluorescentprotein fused to the N-terminus of Bfl-1.

Bim ⁄ Bod exerts its effects by interacting with anti-apoptotic Bcl-2 family members such as Bcl-2 and Bcl-xL. There are three functional splice variants: Bim-Long, Bim-Short and Bim-Extra Long. In this study, theexpression of the Bim protein was examined by using

anti-Bim ⁄ Bod and anti-Bim-Long antibodies. AlthoughBim ⁄ Bod is a pro-apoptotic protein, our results dem-onstrated that increased expression of Bim ⁄ Bod wasassociated with perineural infiltration. Bim ⁄ Bod isregulated by post-translational mechanisms that maymaintain the protein in an inactive complex that isunable to interact with anti-apoptotic proteins.37

Increased expression of Bim-Long was associated withmoderately to well-differentiated tumours. The OS ratewas marginally significantly better in patients with lowexpression of Bim-Long and multivariate analysissuggested that expression of Bim-Long could be anindependent prognostic factor for OSCC patients.Although expression of the Bim protein has not yetbeen evaluated in OSCC, one study did find that bothBim and PUMA can provide prognostic information forstage II and III colonic cancer patients receiving 5-fluorouracil-based adjuvant chemotherapy.38

Bid is a p53 effector and its gene is transcriptionallyregulated by p53 in response to c-irradiation. Chemo-sensitivity of cells to the DNA-damaging agents adri-amycin and 5-fluorouracil is critically dependent onthe presence of p53 and the cleavage of Bid. Cleavage ofBid facilitates a conformational change in the mito-chondria-associated Bax, which functions to initiatemitochondrial dysfunction and cytochrome c release.39

In this study, we observed that increased expression ofBid was associated with moderately to well-differenti-ated tumours. There is no other study regarding Bidexpression and OSCC.

Furthermore, we observed that increased expressionof Bax was associated with absence of vascularinvasion and with tumours occurring in the oraltongue. Although Bax is a pro-apoptotic protein,studies have demonstrated that its high expressioncould be associated with a poorer prognosis for oral andovarian cancer.40,41 On the other hand, Xie et al.11

demonstrated that patients with OSCC of the tonguewith high Bax expression had improved survivalcompared with those with low Bax expression.

Bad is a pro-apoptotic Bcl-2 family member andphosphorylation of the Bad protein inhibits its pro-apoptosis function. In our study, we found thatexpression of Bad in OSCC was not significantlyassociated with any of the clinicopathological para-meters analysed. Iwase et al.42 have shown thatexpression of Bcl-2, cellular inhibitor of apoptosisprotein-1 (cIAP-1) and X-linked IAP was down-regu-lated, and Bax was up-regulated by phosphoinositide3-kinase (PI3K) inhibitors, while Bcl-xL, Bak and cIAP-2 expression levels were not affected. They also foundthat Bad phosphorylation was down-regulated by PI3Kinhibitors, suggesting that inhibition of PI3K enhances



the susceptibility of OSCC cells to anticancer drug-mediated apoptosis through regulation of expressionand post-translational modification of both pro- andanti-apoptotic proteins.

PUMA is a p53 target that is required for apoptosisinduced by p53 and various chemotherapeutic agents.Increased expression of PUMA was associated with theabsence of vascular invasion, tumours occurring in thefloor of mouth, and with recurrence. Furthermore, theDFS rate was significantly better in patients with lowexpression of PUMA. Multivariate analysis suggestedthat expression of PUMA could be an independentprognostic factor for OSCC patients. Sun et al.43 foundthat the absence of PUMA activation in head and necksquamous cell carcinoma (HNSCC) cells contributes tochemoresistance and suggested that gene therapy withPUMA might be an efficient substitute for p53to enhance the responses of HNSCC cells to chemo-therapy.

Our results demonstrated that Bak expression inOSCC was associated with the absence of vascularinvasion. Moreover, OS rate was significantly better inpatients with high expression of Bak and DFS rate wasmarginally significantly better in patients with highexpression of Bak. Schoelch et al.10 demonstrated thatBak and Bcl-x proteins were prominently expressed inpremalignant oral lesions and in OSCCs, suggestingdysregulation of normal apoptotic function. Moreover,Bcl-x was expressed in all stages of disease, andcorrelated with Bak expression. Xie et al.44 demon-strated that Bak expression, particularly in combina-tion with Bax, as well as in combination with apoptoticindex or p53 accumulation, had prognostic value intongue SCC.

The discordance amongst the different studies and thelack of strong predictive power of new tissue biomar-kers, questioning their clinical usefulness as universalpredictors of clinical outcome of patients with HNSCC,may be due to different causes: variations in the locationof the primary tumour site, the sensitivity of thetechnique used for specimen study, the antibodyselected for immunohistochemistry, the quality of thespecimen studied (frozen or paraffin-embedded), han-dling of specimens for immunohistochemistry, storage,fixation, staining protocols and the arbitrary cut-offvalues for positivity of immunoreactivity. More impor-tantly, lack of uniform curative protocols throughoutthese studies may significantly affect their outcome, asthe choice of treatment modality may have a greatimpact on long-term survival of patients with HNSCC.45

Some histopathological features have statisticallysignificant prognostic implications, at least in certainsites of the head and neck: tumour grade, histological

pattern of tumour invasion within the stroma, maxi-mum depth or thickness of tumour invasion, vascularinvasion an perineural invasion by tumour.45 In thepresent study, vascular invasion and perineural infil-tration were found to be predictive factors for OSCCoutcome. We also found decreased expression of Bcl-xL,Bcl-2-related protein A1, Bak, Bax and PUMA associ-ated with vascular invasion and increased expression ofBim ⁄ Bod and BAG-1 associated with perineural infil-tration, suggesting that these proteins might play animportant role in OSCC progression.

In summary, Bcl-xL, Bak, Bax and Bim-Long were allprominently expressed, Bcl-2 was occasionally ex-pressed and Bcl-x, Bcl-2-related protein A1, BAG-1,Bim ⁄ Bod, Bad, Bid and PUMA expression was variablein OSCC samples. Our results have demonstrated thatBcl-2, Bcl-x and Bad proteins were not significantlyassociated with any of the clinicopathological charac-teristics analysed. Increased expression of Bak, Bax,Bcl-xL, Bcl-2-related protein and PUMA was associatedwith the absence of vascular invasion in OSCC.Increased expression of Bax was also associated withtumours occurring in the oral tongue. Increasedexpression of Bim ⁄ Bod and BAG-1 was associated withthe presence of perineural infiltration. Increased expres-sion of Bid and Bim-Long was associated with moder-ately to well-differentiated tumours. Increasedexpression of Bcl-2-related protein A1 was associatedwith early-stage tumours and with tumours occurringin the floor of the mouth. Increased expression of PUMAwas also associated with tumours occurring in the floorof the mouth and with tumour recurrence. MultivariateCox analysis demonstrated that PUMA and Bim-Longwere independent factors in prognosis of OSCC.

The contradictory results concerning prognosis andexpression of Bcl-2 family proteins in this study indicatethat induction of apoptosis is very complex and theinfluence of individual proteins may vary from tumourto tumour. It is also important to note that it is thebalance between anti- and pro-apoptotic proteins thatdetermines the cell fate. We have established groups ofcases with patterns of anti- and pro-apoptotic expres-sion; however, we found no association between thesepatterns and OS. It is important to note that theseproteins can be diversely regulated by post-transla-tional modifications, such as phosphorylation or pro-teolytic cleavage, and also the balance betweenapoptotic and survival signalling pathways may bethe result of apoptosis-regulating miRNAs.

In conclusion, the present study provides evidence forthe involvement of the Bcl-2 family of proteins in OSCCtumorigenesis. To our knowledge, this is the first studyto evaluate such a large panel of Bcl-2 family members



and suggests that the expression of apoptotic moleculesmight be used as a prognostic indicator for OSCC.

Acknowledgements

FAPESP 98 ⁄ 14335-2 and 07 ⁄ 50608-4.

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Expression of Bcl-2 family proteins and associated clinicopathologic factors predict survival...

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