Correlation Among Six Biologic Factors (p53, p21WAF1, MIB-1, EGFR, HER2, and Bcl-2) and Clinical...

Int. J. Radiation Oncology Biol. Phys., Vol. 74, No. 4, pp. 1165–1172, 2009Copyright � 2009 Elsevier Inc.

Printed in the USA. All rights reserved0360-3016/09/$–see front matter

doi:10.1016/j.ijrobp.2008.09.005

CLINICAL INVESTIGATION Cervix

CORRELATION AMONG SIX BIOLOGIC FACTORS (P53, P21WAF1, MIB-1, EGFR, HER2,AND BCL-2) AND CLINICAL OUTCOMES AFTER CURATIVE CHEMORADIATION

THERAPY IN SQUAMOUS CELL CERVICAL CANCER

HIDEOMI YAMASHITA, M.D., PH.D., NAOYA MURAKAMI, M.D., TAKAO ASARI, M.D., KAE OKUMA, M.D.,KUNI OHTOMO, M.D., PH.D., AND KEIICHI NAKAGAWA, M.D., PH.D.

Department of Radiology, University of Tokyo Hospital, Tokyo, Japan

Purpose: The expressions of six cell-cycle–associated proteins were analyzed in cervical squamous cell carcinomasin correlation in a search for prognostic correlations in tumors treated with concurrent chemoradiation therapy(cCRT).Methods and Materials: The expressions of p53, p21/waf1/cip1, molecular immunology borstel-1 (MIB-1), epider-mal growth factor receptor (EGFR), human epidermal growth factor receptor type 2 (HER2), and Bcl-2 were stud-ied using an immunohistochemical method in 57 cases of cervical squamous cell carcinoma treated with cCRT.Patients received cCRT between 1998 and 2005. The mean patient age was 61 years (range, 27–82 years). The num-ber of patients with Stage II, III, and IVA disease was 18, 29, and 10, respectively.Results: The number of patients with tumors positive for p53, p21/waf1/cip1, MIB-1, EGFR, HER2, and Bcl-2 was26, 24, 49, 26, 13, and 11, respectively; no significant correlation was noted. The 5-year overall survival rates ofHER2-positive and -negative patients was 76% vs. 44%, which was of borderline significance (p = 0.0675). Nosignificant correlation was noted between overall survival and expressions of p53, p21/waf1/cip1, MIB-1,EGFR, and Bcl-2. No correlation was observed between local control and expression of any of the proteins.Conclusion: Expression of HER2 protein had a weak impact of borderline significance on overall survival in squa-mous cell carcinoma of the uterine cervix treated with cCRT. However, no clinical associations could be establishedfor p53, p21/waf1/cip1, MIB-1, EGFR, and Bcl-2 protein expressions. � 2009 Elsevier Inc.

Uterine cervix, Immunohistochemistry, Squamous cell carcinoma, Prognostic factors, Biologic markers.

INTRODUCTION

Cervical adenocarcinoma treated with radiation therapy (RT)

alone has been studied by various investigators searching for

information that could be correlated with prognosis. A recent

immunohistochemical study showed that progesterone recep-

tor status was associated with a favorable prognosis (1).

Tsuda et al. (2) reported that overexpression of the p53

gene product correlated with a poor prognosis. A study by

Kihara et al. (3) showed that overexpression of the c-erbB2

proto-oncogene product was greater in patients with ad-

vanced clinical stage disease and indicated a poor prognosis.

Lu et al. (4) reported that expression of the p21/waf1/cip1

product correlated with a favorable prognosis. The cell-

cycle–associated proteins p53, p21/waf1/cip1, molecular

immunology borstel-1 (MIB-1), epidermal growth factor re-

ceptor (EGFR), human epidermal growth factor receptor

type 2 (HER2) and Bcl-2, commonly reported as prognostic

factors in many other sites, were the subjects of this immuno-

histochemical study. This study was conducted on biopsy

Reprint requests to: Dr. Keiichi Nakagawa, Department of Radi-ology, University of Tokyo Hospital, 7-3-1, Hongo, Bunkyo-ku,Tokyo, 113-8655, Japan. Tel: (+81) 3-5800-8667; Fax: (+81)3-5800-8935; E-mail: [email protected]

11

specimens excised from patients before cCRT to elucidate

the effects of RT with respect to prognostic significance.

METHODS AND MATERIALS

Patients and specimensThis study involved 57 consecutive patients with squamous cell

carcinoma (SCC) of the uterine cervix, who received concurrent che-

moradiation therapy (cCRT) alone at the University of Tokyo Hospi-

tal (Tokyo, Japan). All patients without distant metastasis between

1998 and 2005 who had paraffin-embedded tumor samples were stud-

ied. The median patient age was 61 years (range, 27–82 years). Clin-

ical stage and histologic classification were based on the criteria

established by the International Federation of Gynecology and Obstet-

rics (FIGO) (5) and the World Health Organization (6). The number of

patients with FIGO Stage II, III, and IVA disease was 18, 29, and 10,

respectively. Histologically, all patients had been diagnosed with

squamous cell carcinoma. The median follow-up period was 30.9

months (range, 4–121 months). Twenty-nine deaths were attributed

to the cancer. Five patients died of other diseases, and none were

lost to follow-up. The surviving 23 patients were followed for

Conflict of interest: none.Received Aug 6, 2008, and in revised form Sept 6, 2008.

Accepted for publication Sept 10, 2008.

65

mailto:[email protected]

1166 I. J. Radiation Oncology d Biology d Physics Volume 74, Number 4, 2009

a median of 66.4 months (range, 27–121 months). All biopsy speci-

mens were excised from the cervical tumors before cCRT, fixed in

10% formalin for 24 h, and embedded in paraffin. Clinical character-

istics and follow up of patients are summarized in Table 1.

RadiotherapyPatients were treated with a combination of conventional pelvic

external beam cCRT and high-dose-rate intracavitary cCRT. The

details of the treatment protocol have been previously reported

(7–9). External beam cCRT energy was 10 MV. A four-field irradi-

ation technique was used to spare the small intestine. Treatment po-

sition was supine. Simulation was by computed tomography (CT)

simulator. The standard treatment protocol of our institution consists

of the following: whole-pelvis radiotherapy (WPRT) of 19.8 Gy in

11 fractions (Fr) plus WPRT with a midline block (MB) of 30.6 Gy

in 17 Fr for all Stage IIA and Stage IIB without a bulky mass

Table 1. Patient characteristics (N = 57)

Characteristic N (%)

AgeMedian (range) 61 (27–82)<35 2 435–54 15 26$55 40 70

FIGO stageII 18 32III 29 50IVA 10 18

HistologySquamous 57 100

Histologic typeKeratinizing 24 48Nonkeratinizing 26 52

Follow-up interval (mo)median (range)All patients 30.9 (4.0–120.8)Alive patients 66.4 (27.4–120.8)

Follow-upAlive 23 40Death from carcinoma 29 51Death from unrelated disease 5 9

Abbreviation: FIGO = International Federation of Gynecologyand Obstetrics.

($4cm); WPRT of 30.6 Gy in 17 Fr plus WPRT with MB of 19.8

Gy in 11 Fr for Stage IIB with a bulky mass all Stage IIIA and Stage

IIIB without a bulky mass; and WPRT of 39.6 Gy in 18 Fr plus

WPRT with MB of 10.8 Gy in 6 Fr for Stage IIIB with a bulky

mass and Stage IVA. Patients treated postoperatively were ex-

cluded. In addition to central shielding cCRT, these patients also

underwent intracavitary cCRT. Theintracavitary cCRT was per-

formed with Ir-192 sources with four insertions (one or two weekly),

at fraction doses of 6.0 Gy at Point A. Concurrent chemotherapy was

combined with cCRT in all patients. All patients received cisplatin-

alone (75 mg/m2 in a bolus infusion on Days 1, 22, and 43).

ImmunohistochemistryThe characteristics of the primary antibodies in immunohistochem-

istry are summarized in Table 2. The level of expression was assessed

semiquantitatively using the immunoreactive scoring (IRS) system.

The IRS value was determined by considering the intensity, graded

on a scale of 0–3 (0 = no staining, 1 = weak staining, 2 = moderate

staining, and 3 = strong staining) and extent (percentage of positive

tumor cells) of staining. The extent of staining was graded on a scale

of 0–4 (0 = no staining; 1 = 1–25% staining, 2 = 26–50% staining, 3 =

51–75% staining, and 4 = 76–100% staining). An IRS value (range,

0–7) was obtained by adding the two parameters. The cutoff level of

expression of the biochemical markers was defined as a dichotomous

variable of low expression (IRS value 0–5) or high (IRS 6–7) in p53,

p21, MIB-1, and Bcl-2; low (0–4) or high (5–7) in EGFR; and low

(0–3) or high (4–7) in HER2, respectively (Table 3).

Statistical analysisAssociations between variables were assessed using the Chi-

square, Fisher exact, and linear-by-linear exact tests. The Kaplan-

Meier product-limit method was used to estimate the probability of

overall survival, disease-free survival, and locoregional recurrence-

free survival; the log–rank test was used to estimate any

differences. Multivariate analyses were performed using the Cox pro-

portional hazards regression model. Overall survival was calculated

in months from the first day of cCRT to the date of death from any

cause or to February 2008. Patients who were still alive in

February 2008 were treated as censored. Values of p < 0.05 were

regarded as statistically significant. Statistical analyses were per-

formed using StatView Dataset File version 5.0 J for Windows com-

puters (SAS Institute, Cary, NC).

Table 2. Immunohistchemistry: characteristics of the primary antibodies

Protein Type Source Pretreatment Titer Incubation Control Staining

p53 Mouse MC DO-7Novocastra

Microwave 1 mmol/lEDTA (pH8.0)

1:200 30 min 37�C Breastcancer

Nucleus

p21 Mouse MC SX118DakoCytomation


1:25 30 min 37�C Breastcancer

Nucleus

MIB-1 Mouse MC MIB-1DakoCytomation


1:25 30 min 37�C Intestine Nucleus

HER2 Rabbit PC -DakoCytomation

Water bath (99�C)Citrate buffer (pH6.0)

R-to-U 30 min RT Breastcancer

Membrane

EGFR Mouse MC 2-18C9DakoCytomation

Proteinase K (kit) R-to-U 30 min RT HT-29(cells)

Membrane(Cytoplasmic)

bcl-2 Mouse MC 124DAKO

Microwave 1 mMEDTA (pH8.0)

1:80 30 min 37�C Lymphoma Cytoplasmic

Abbreviations: RT = room temperature; R-to-U = ready to use (kit).

Immunohistochemical study of cervical squamous cell carcinoma d H. YAMASHITA et al. 1167

RESULTS

Staining results in SCC cases are summarized in Tables 3

and 4. Table 4 shows the positive rates of p53, p21/waf1/cip1,

MIB-1, EGFR, HER2, and Bcl-2 according to patient age,

T-stage, N-stage, paraaortic lymph node metastasis, and

tumor size. Representative staining results are illustrated in

Figure 1. The positivity of MIB-1 was greater for larger tumor

size. Similarly, the positivity of Bcl-2 was greater in older

patients. However, no significant correlation was detected

in the expression of the proteins studied (Table 4).

The 5-year overall survival rates of the positive and nega-

tive factors, respectively, were as follows: p53, 48% vs. 51%;

p21/waf1/cip1, 54% vs. 42%; MIB-1, 44% vs. 49%; EGFR,

46% vs. 52%; HER2, 44% vs. 76%; and Bcl-2, 47% vs. 57%.

The overall survival rates of HER2-positive and -negative

patients was of borderline significance (p = 0.0675)

(Fig. 2). No statistically significant difference was found

among the positive and negative groups in the five other pro-

teins (p53, p = 0.4832; p21, p = 0.6786; MIB-1, p = 0.9420;

EGFR, p = 0.8291; and Bcl-2, p = 0.9799). Even when limit-

ing the analysis to the 47 patients with Stages II and III disease

(i.e., excluding 10 patients with Stage IVA), no significant

difference was found among the positive and negative groups

in the all of the proteins including HER2 (Table 5).

With regard to disease-free survival, no significant differ-

ence was found among the positive and negative groups for

all six proteins (p53, p = 0.3515; p21, p = 0.8947; MIB-1,

p = 0.3838; EGFR, p = 0.7625; HER2, p = 0.1377; and Bcl-

2, p = 0.2155). Moreover, for locoregional (i.e., within-pelvis)

recurrence-free survival, no significant difference was found

among the positive and negative groups for all six proteins

(p53, p = 0.8984; p21, p = 0.9018; MIB-1, p = 0.3016;

EGFR, p = 0.9949; HER2, p = 0.2321; and Bcl-2, p = 0.9555).

Multivariate analysis by Cox regression showed that

lymph node metastasis and FIGO staging were powerful

Table 3. Staining results in all patients

Variable N (%) 5-Year OS p Value

p53Negative (0–5) 31 54 48% 0.4832Positive (6, 7) 26 46 51%

p21Negative (0–5) 33 58 54% 0.6786Positive (6, 7) 24 42 42%

MIB-1Negative (0–5) 8 14 44% 0.9420Positive (6, 7) 49 86 49%

EGFRNegative (0–4) 31 54 46% 0.8291Positive (5–7) 26 46 52%

bcl-2Negative (0–5) 46 81 47% 0.9799Positive (6, 7) 11 19 57%

HER2Negative (0–3) 44 76 44% 0.0675Positive (4–7) 13 24 76%

Abbreviation: OS = overall survival.

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Fig. 1. Immunohistochemical analysis of biospy specimens from squamous cell carcinoma patients showing high (a) p53in Case 29, (b) p21 in Case 26, (c) molecular immunology borstel-1 (MIB-1) in Case 14, (d) epidermal growth factor re-ceptor (EGFR) in Case 30, (e) human epidermal growth factor receptor type 2 (HER2) in Case 20, and (f) Bcl-2 in Case 23.Original magnification �200.

predictors for the survival of patients, whereas HER2-posi-

tivity was not significant (Table 6).

DISCUSSION

In this study, a set of cervical cancer patients treated with

cCRT were retrospectively assessed for a series of molecular

markers versus outcome. The cohort was obtained over 1998

to 2005 and consisted of 57 cases of cervical SCC treated

with cCRT. This group included patients in disease Stages

II to IV. This is the first report evaluating six biomarkers con-

curently as prognostic factors in the same population.

The p53 gene, present on chromosome 17p, acts as a tumor

suppressor gene, controlling entry into the S phase of the cell


0

20

40

60

80

100

Overall su

rvival rate (%

)

0 20 40 60 80 100 120 140

Months

Kaplan-Meier method

HER2(+) (n=9)

HER2(-) (n=29)

Log-rank p=0.0675

HR=3.581 (95%CI:0.828-15.481)

(a)

0

20

40

60

80

100

Overall su

rvival rate (%

)

0 20 40 60 80 100 120 140

Months

Kaplan-Meier method

Stage I-III (n=47)

Stage IV (n=10)

Log-rank p<0.0001

HR=0.221 (95%CI:0.101-0.484)

(b)

0

20

40

60

80

100

OS

rate (%

)

0 20 40 60 80 100 120 140

Months

Kaplan-Meier method

PLN(-) (n=48)

PLN(+) (n=9)

Log-rank p=0.0008

HR=0.257 (95%CI:0.110-0.604)

(c)

0

20

40

60

80

100

OS

rate (%

)

0 10 20 30 40 50 60 70

Months

Kaplan-Meier method

PALN(-) (n=44)

PALN(+) (n=13)

Log-rank p=0.0007

HR=0.298 (95%CI:0.141-0.626)

(d)

Fig. 2. Survival curves for squamous cell carcinoma patients (a) with or without human epidermal growth factor receptortype 2 (HER2) overexpression, (b) between International Federation of Gynecology and Obstetrics Stage I–III and StageIV, (c) with or without pelvic lymph node metastasis, (d) with or without paraaortic lymph node metastasis. p Values wereobtained from log-rank tests.

Table 5. Staining results in 46 patients with Stage II or IIIdisease

N 5-year OS p Value

p53Positive 19 67% 0.9174Negative 27 50%

p21Positive 17 58% 0.6285Negative 29 58%

MIB-1Positive 26 59% 0.9174Negative 20 43%

EGFRPositive 20 63% 0.8569Negative 26 53%

HER2Positive 12 88% 0.2230Negative 34 49%

Bcl-2Positive 10 57% 0.4775Negative 36 57%

Abbreviation: OS = overall survival.

cycle. Wild-type p53 activates p21 and induces G1 arrest in

the cell cycle (10). Mutation of this gene inactivates this tu-

mor suppressor activity and is related to tumor progression

and survival (11). A correlation between p53 gene mutation

and unfavorable outcome in cervical cancer had been re-

ported (12). Oka et al. (13) reported that p53 overexpression

has a significant correlation with poor outcome of cervical

SCC. Tsuda et al. (2) found that p53 overexpression in

Table 6. Cox multivariate regression analysis in patientswith squamous cell of the cervix carcinoma

Variable RR 95% CI p Value

HER2: negative 2.180 0.451–10.534 0.3322PLN: negative 0.765 0.224–2.605 0.6679PALN: negative 0.333 0.098–1.134 0.0785Age $60 years 0.727 0.235–2.245 0.5791FIGO stage 0.0326

II 0.376 0.103–1.372 0.1386III 0.280 0.071–1.098 0.0679

Abbreviations: CI = confidence interval; FIGO = InternationalFederation of Gynecology and Obstetrics; RR = relative risk; PLN =pelvic lymph node; PALN = paraaortic lymph node.


adenocarcinoma of the cervix had a statistically significant

correlation with poor outcome. Conversely, Lu et al. (4)

reported that p53 expression was not correlated with the

prognosis of cervical adenocarcinoma. In our study, p53

overexpression has no significant association with outcome

of cervical SCC treated with cCRT.

The p21/waf1/cip1 gene, located on chromosome 6p,

encodes a protein that inhibits a variety of cyclin-cyclin–

dependent kinase (CDK) complexes (14). The p53 control

of cell proliferation is mediated predominantly through

p21/waf1/cip1, the transcription of which is induced by

wild-type p53. The substance p21/waf1/cip1 triggers G1 ar-

rest or apoptosis in cells with the wild-type p53 gene (10).

Niibe et al. (15) reported that overexpression of p21/waf1/

cip1 correlated with p53 overexpression and apoptosis during

cCRT for cervical SCC. In our study, no correlation was ob-

served between the overexpression of p21/waf1/cip1 and

p53. Lu et al. (4) reported that overexpression of p21/waf1/

cip1 was a favorable prognostic factor in surgically treated

cervical adenocarcinoma. The present results indicated that

no significant correlation exists between p21 status and prog-

nosis in cervical SCC treated by cCRT.

Ki-67/MIB-1 monoclonal antibody recognizes proliferating

cells in the G1, S, G2, and M phases of the cell cycle (16). The

relationship between Ki-67/MIB-1 labeling index and con-

ventional prognostic parameters and survival is controversial

in cervical cancer (17–27). The present study also indicated

that no statistically significant correlation exists between

MIB-1 status and prognosis in cervical SCC treated by cCRT.

The Bcl-2 gene family seems to act as a regulator of the ap-

optotic pathway (28–30). The two most important apoptosis

regulating proteins of this family are most likely Bcl-2 and

bax. Bcl-2 is a member of the anti-apoptotic family (31). It

probably acts as a rheostat for the cell death program (29,

31). Generally, an overexpression of Bcl-2 would prevent ap-

optosis of the cell, without promoting cell proliferation or cell

cycle progression. The present study did not show a signifi-

cant role for Bcl-2 expression in cervical cancer.

Both EGFR and HER2 have been shown in some studies to

be independent predictors of a poor prognosis in cervical car-

cinoma (32–39). However, other studies have also revealed no

significant association with an adverse outcome (32, 40–43).

Although conflicting results have been reported, it is notewor-

thy that in the analysis by Lee et al. (44) HER2 overexpression

was a favorable prognostic factor in both univariate and multi-

variate analyses for OS. After controlling for pretreatment fac-

tors (stage, grade, and histology) in their model, multivariate

analysis of all biomarkers revealed that only increased expres-

sion of HER2 was associated with improved OS (hazard ratio,

0.31, 95% confidence interval, 0.10–0.99, p = 0.05). Leung

et al. (40) found that HER2 played a more significant role in

tumor progression and invasion for cervical carcinoma than

EGFR, but neither HER2 nor EGFR was found to carry any

prognostic significance. Ngan et al. (41) also described the

lack of correlation between clinical prognosis and HER2

expression. In other previous studies (42), elevated HER2neu

has been correlated with tumor size, local failure, and dimin-

ished survival in cervical cancer. In our study, HER2 overex-

pression had no correlation with tumor size or overall

survival. Kihana et al. (37) found in their analysis of 44 patients

with cervical adenocarcinoma that increased expression of

HER2 correlated with a poorer prognosis. In the majority of

these previous clinical studies, adenocarcinomas were evalu-

ated. In our study population, all of the tumors consisted of

squamous cell histology and may account for the differences

in biomarker correlates. The present study is the first report

to show a correlation of borderline significance between

HER2 overexpression and unfavorable outcome in patients

with cervical SCC treated with cCRT.

Given the conflicting findings in literature noted above, larger

sample sizes may help to resolve definitively the issue of

whether these six proteins can serve as correlates for outcomes.

In this study, only 11 patients scored positive for bcl-2 expres-

sion, and only 13 patients scored positive for HER2 expression.

This study was limited to the analysis of six proteins

previously reported on in other studies separately. Because

the lack of correlation with these six proteins was noted, other

biologic correlates may be tested. For example, because

correlation of X-ray repair cross-complementing group 1 pro-

tein and cisplatin therapy in a variety of cancers (45–47) has

been reported, this may be a candidate biologic marker to

study.

CONCLUSION

In conclusion, we investigated the tumor expression of

several proteins involved in cell cycle control and associated

with cell proliferation and cell division. HER2 protein ex-

pression was shown to have a weak impact on the prognosis

of patients with cervical SCC treated with cCRT. Clinical sig-

nificance of p53, p21/waf1/cip1, MIB-1, EGFR, and Bcl-2

protein expression was not found in cervical SCC.

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Correlation Among Six Biologic Factors (p53, p21WAF1, MIB-1, EGFR, HER2, and Bcl-2) and Clinical...

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