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ABSTRACT

Introduction: Anthracyclines repre-

sent one of the most important

chemotherapeutics in breast cancer.

However, they cause cardiac damage

besides some tumors might be anthra-

cycline-resistant.

Aim of the work: The aim of the pre-

sent work was to study the predictive

value of human epidermal growth fac-

tor receptor 2 (HER2/neu) protein,

state (positive or negative) and score

(0,1+,2+,3+), for the outcome of 5-

fluorouracil / Adriamycin / cyclophos-

phamide (FAC) adjuvant chemother-

apy in Egyptian high risk female

breast cancer patients.

Subjects & Methods: The present ret-

rospective cohort study was conducted

in Alexandria Main University Hospi-tal, Egypt. It included fifty high risk

female breast cancer patients

(according to St Gallen guidelines

2007) with operable breast cancer. All

of them have received FAC adjuvant

chemotherapy between January 2007

and December 2007 and were followed

for 2 years. Pretreatment breast tu-

mor samples were obtained from for-

malin fixed/paraffin-embedded tissue

blocks where HER2/neu protein was

assessed by immunohistochemistry

(IHC).

Results: Kaplan-Meier survival analy-

sis showed that positive HER2/neustate was associated with superior 2-

year disease free survival (DFS) and

overall survival (OS) rates to negative

HER2/neu state but this difference

was statistically insignificant (log rank

p=0.08 and 0.24 respectively). On the

other hand, there was a statistically

significant difference between 2-year

DFS rate of different HER2/neu pro-

tein score categories (log rank

p=0.001*). Although being statistically

significant, there was no pattern be-

tween HER2/neu protein scoring andDFS where score 0 showed intermedi-

ate DFS rate between scores 2+ and

3+. Moreover, Cox regression analysis

showed that HER2/neu protein score

couldnt be an independent predictor

for the outcome (DFS) of FAC adju-

vant chemotherapy (p=0.66).

THE PREDICTIVE VALUE OF HUMAN EPIDERMAL GROWTH FACTOR

RECEPTOR 2 (HER2/neu) FOR THE OUTCOME OF ANTHRACYCLINE-

BASED ADJUVANT CHEMOTHERAPY IN EGYPTIAN HIGH RISK FEMALE

BREAST CANCER PATIENTS

Ahmed N. Abd El-Aal1, Nashaat S. Lotfy

2, Ehsan M. H. Abd Al-Rahman

1,

Suzan M. F. Helal3, Eman M. S. Kamha

1, Doaa A. Abd Al-Monsif

1,

Mohammed S. Shaat4, Ahmed S. Anan4.Departments of Medical Biochemistry1, Clinical Oncology and Nuclear Medicine2,

Pathology3, andUndergraduate students

4, Faculty of Medicine,

Alexandria University, Egypt.

THE EGYPTIAN JOURNAL OF MEDICAL SCIENCES VOL. 32-No. 1 JUNE 2011 (ISSN: 1110-0540)

8. Egypt. J. Med. Sci. 32 (1) June 2011: 107-118.


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Conclusion: HER2/neu protein might-

nt be considered as a predictor for the

outcome of FAC adjuvant chemother-

apy in Egyptian high risk female

breast cancer patients. Consequently,

large-scale prospective studies will be

needed to clearly define its predictive

value.

INTRODUCTION

Breast cancer is the most com-

monly diagnosed cancer in womenworldwide and is the second leading

cause of deaths in women after lung can-

cer (Jemal et al., 2009). In Egypt, breast

cancer is the most prevalent cancer

among women where it constitutes 29%

of the National Cancer Institute cases

(Omar et al., 2003). Breast cancer, like

all malignancies, arises as a result of the

accumulation of genetic alterations, espe-

cially deregulation of the expression ofoncogenes and tumor suppressor genes

(Omar et al., 2003). One of the most im-

portant adjuvant chemotherapeutics usedin breast cancer are anthracycline-based

combinations (Buzdar, 2006). A major

component of anthracycline cytotoxicity

is due to topoisomerase II (TOPOII) poi-

soning. Moreover, they intercalate into

double-stranded deoxyribonucleic acid

(dsDNA) and produce structural changes

that interfere with DNA and ribonucleic

acid (RNA) synthesis. Besides, anthracy-

clines generate reactive oxygen species

(ROS) that damage DNA, messenger

RNA (mRNA), proteins and lipids; the

peroxidation of lipids may account formuch of the cardiac toxicity characteris-

tic of these drugs (Rubin and Hait, 2003).Several predictive factors that

might affect the outcome of chemother-

apy in breast cancer were studied but

none of them was proved to have enough

predictive power (Tewari et al., 2008).

Consequently, the choice of chemothera-

peutic drugs in breast cancer is usually

empiric and 30%70% of patients with

measurable disease fail to respond

(Kennedy et al., 2004). Additionally,

financing cancer treatment is a major

challenge especially for developing coun-

tries (Boutayeb et al., 2010). From here

comes the importance of predictive fac-

tors in tailoring of chemotherapy.The human epidermal growth factor

receptor 2 gene is localized to chromo-some 17q11.2-12. It encodes a 185 kDa

transmembrane tyrosine kinase receptor

that is a member of the epidermal growth

factor receptor (EGFR) family (Siddig etal., 2008). These proteins possess an ex-

tracellular ligand-binding domain, a

membrane-spanning region and a cyto-

plasmic domain with tyrosine kinase ac-

tivity (Duffy, 2005). After heterodimeri-

zation, HER2/neu complexes initiate

intracellular signaling via the Ras/

mitogen-activated protein kinase

(MAPK) pathway, the phosphatidylinosi-

tol 3-kinase (PI3K) pathway, the Janus

kinase (JAK)/signal transducer and acti-vator of transcription (STAT) pathway

and the phospholipase C-gamma (PLC-)

pathway. Activation of the latter path-

ways ultimately promote cell prolifera-

tion, survival, motility and adhesion

(Duffy, 2005). HER2/neu is overex-pressed in 15-25% of patients with breast

cancer and was reported to be an unfa-

vorable prognostic factor in breast cancer

(Tewari et al., 2008). Furthermore, the

possible relation between HER2/neu

overexpression and the outcome of an-

thracycline-based regimens in breast can-

cer was hypothesized (Zhang and Liu,

2008).

The aim of the present work was to

reveal the possible predictive value ofHER2/neu protein, state (positive or

108 Abd El-Aal et al.

Egypt. J. Med. Sci. 32 (1) 2011


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negative) and score (0, 1+, 2+, 3+), for

the outcome of anthracyclinebased ad-

juvant chemotherapy in Egyptian high

risk female breast cancer patients. The

predictive value was assessed in terms of

disease free survival (DFS), overall sur-

vival (OS) as well as time of relapse and

type of relapse (local or distant). DFS

was defined as the time between the date

of randomization and the date of diseaserelapse and OS was defined as the time

between randomization and death from

any cause. Cardiotoxicity from anthracy-

cline and mortality were recorded as

well.

SUBJECTS & METHODS

The present retrospective cohort

study was conducted in Alexandria Main

University Hospital, Egypt. It included

fifty high risk female breast cancer pa-

tients according to St Gallen guidelines2007 (Goldhirsch et al., 2007). Patients

were randomly recruited from the Clini-cal Oncology Department. All of them

have received anthracyclinebased

(FAC) adjuvant chemotherapy between

January 2007 and December 2007. Pre-treatment breast tumor samples were

obtained from formalin fixed/paraffin-

embedded tissue blocks, in accordance

with the standard methods, from the ar-

chives of the Pathology Department.

Before starting the treatment, all patientsunderwent a complete physical examina-

tion, laboratory and radiological investi-

gations. Diagnosis was confirmed by afine needle aspiration (FNA) or a core

biopsy of the primary tumor. Modified

radical mastectomy was the standard line

of treatment. After surgery, all patients

received 6 cycles of FAC and postmas-

tectomy irradiation. In addition, hormone

receptors [estrogen receptors (ER) and/or

progesterone receptors (PR)] positive

cases were assigned to receive tamoxifen

for a period of 5 years. After finishing

FAC-adjuvant chemotherapy, patients

were followed every 2 months for a pe-

riod of 2 years to detect local recurrence

or distant metastasis. In addition, cardio-

toxicity from anthracycline and mortality

were recorded. During the follow-up

period, patients were subjected to: thor-ough clinical examination, laboratory

investigations, periodic radiological ex-

amination, ejection fraction and electro-

cardiogram (ECG) when needed. An

informed consent was taken from every

patient and the study was approved bythe Institutional Ethics Committee. All

breast cancer tumor samples were sub-

jected to full histopathological examina-

tion. ER and PR states were determined

as well (Tafjord et al., 2002). Further-

more, breast cancer tumor samples were

subjected to the following assay:

Immunohistochemical (IHC) stainingprotocol of HER 2/neu protein

Human epidermal growth factor

receptor 2 overexpression was assessedby IHC using primary monoclonal anti-

body for HER2/neu [LabVision Corpora-

tion (Neo Markers, Fremont, USA)

http://www.labvision.com]. Expression

of HER2/neu was visualized using the

streptavidin-biotin-immunoenzymaticantigen detection system which was per-

formed in accordance to the manufac-

turer's protocol (Rocha et al., 2009). Posi-tive and negative controls were included

in all runs. Positive HER2/neu immu-

nostaining was defined as brown cell

membrane staining of the breast cancer

tissue. The threshold for Her2/neu was

assessed as described: no staining at all

or membrane staining in


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observed tumor cells was considered

negative (0). A faint/barely perceptible

membrane staining in >10% of tumor

cells or staining of part of their mem-

brane was scored as negative (1+). A

weak to moderate staining of the entire

membrane in >10% of the tumor cells

was considered weakly positive (2+). A

moderate to strong staining of the entire

membrane in >10% of the tumor cellswas scored as strongly positive (3+)

(Orlando et al., 2008). HER2/neu protein

state was classified into negative (score 0

and 1+) and positive (score 2+ and 3+)

(Orlando et al., 2008) (Fig. 1).

Statistical Analysis

Demographic data including diag-

nosis details, tumor characteristics, che-

motherapy scheme, toxicity and outcome

were entered prospectively into a data-

base. Continuous data were expressed asmean (for normally distributed data) or

median (for abnormally distributed data)and range between parentheses. Qualita-

tive data were presented as absolute

numbers and percentages. Survival

curves were obtained by the KaplanMeier method. Differences between the

survival curves were investigated using

the log-rank univariate analysis. A re-

gression analysis based on the Cox pro-

portional hazards model was conducted

using selected covariates to reveal thepossible independent predictors for DFS

and OS. Mann-whitney test was used to

assess the possible effect of HER2/neuprotein on the time of relapse. Chi-

square test was conducted to assess the

possible relation between HER2/neu

protein and the type of relapse. Data

were analyzed using SPSS software

package version 18.0. All statistical tests

were two sided and probability values of

p


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couldnt be an independent predictor for

better DFS after FAC therapy (p=0.66)

(Table 2). Positive HER2/neu protein

state was associated with a superior 2-

year OS rate (83.3%) which was statisti-

cally insignificant from negative HER2/

neu protein state (69.2%) (log rank

p=0.24) (Table 2, Fig. 2B). Furthermore,

there was a statistically insignificant

difference between 2-year OS rate ofdifferent HER2/neu protein score catego-

ries (log rankp=0.31) (Table 2, Fig. 2B).

Among the relapsed patients, HER2/neu

failed to show a significant relation to

the type of relapse (p=0.11) (Table 3).

Moreover, it had no significant effect on

the time of relapse (HER2/neu positive

state: 10 cases, mean=10.95.32 months;

HER2/neu negative state: 18 cases,

mean=12.385.86 months; Mann-

whitneyp=0.48).

Figure 1.

A. Weak cytoplasmic positive stain with no membranous stain of all tumor cells

[(negative score 0) immunostain HER2/neu (X40)].B. faint barely perceptible membrane stain in more than 10% of cells [(negative score 1+)

immunostain HER2/neu (X20)].

C. moderate stain of all membrane in 10% of cells [(weak positive score 2+) immu-

nostain HER2/neu (X40)].

D. strong positive staining of the whole membrane, in all tumor cells [(positive score 3+)

immunostain HER2/neu (X20)].

HER2/neu Value in Anthracycline Therapy in Breast Cancer 111

Egypt. J. Med. Sci. 32 (1) 2011


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Figure 2.

A. Kaplan-Meier disease free survival (DFS) curve for FAC-treated breast cancer casesin Alexandria, Egypt, according to:

A1. HER2/neu protein state.

A2. HER2/neu protein score.

B. Kaplan-Meier overall survival (OS) curve for FAC-treated breast cancer cases in Al-

exandria, Egypt, according to:B1. HER2/neu protein state.

B2. HER2/neu protein score.

Kaplan-Meier curves were plotted in Harvard Graphics.


Egypt. J. Med. Sci. 32 (1) 2011


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Table (1): Clinicopathological features of the studied patients

Variable No. %

Age


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Table (2): Univariate (Log Rank) and multivariate (Cox proportional hazards regression)

analysis for the HER2/neu protein state and score according to: A. disease free survival(DFS) and B. overall survival (OS)

BiomarkerLog Rank Test

Cox Proportional Hazards

Regressiona

p valueb

p value Exp(B)c

A. DFS

HER2/neu Protein State 0.08 - -

HER2/neu Protein Score 0.001* 0.66 -

B. OS

HER2/neu Protein State 0.24 - -

HER2/neu Protein Score 0.31 - -

a. The significant parameters in log rank univariate analysis were included in Cox pro-

portional hazards regression analysis to reveal the possible independent predictorsfor DFS. By log rank test, except for tumor type and ER protein state (log rank

p=0.001* and 0.000* respectively), none of the clinicopathological characteristics of

patients were significantly related to DFS, so only tumor type and ER state were

included in the regression analysis.

b. All statistical tests are two sided and probability (p) value of


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DISCUSSION

Analysis of DFS curve for HER2/neu showed a discrepancy between the

results of survival of HER2/neu protein

state and that of HER2/neu protein score

categories. Additionally, although the

latter was statistically significant, there

was no pattern between HER2/neu pro-tein scoring and DFS as patients with

score 0 had intermediate DFS rate be-

tween scores 2+ and 3+. By Cox regres-sion analysis, HER2/neu protein might-

nt be an independent predictor for better

DFS after FAC therapy. These discrep-ancies could probably be explained by

the relatively small number of patients in

each HER2/neu protein score category

and/or possible IHC fallacies that might

be due to loss of HER2/neu protein as a

result of tissue storage and fixation

(Duffy, 2005). Regarding OS, although

positive HER2/neu protein state was

associated with a superior 2-year OS rate

to negative HER2/neu protein state, thisdifference was statistically insignificant.Besides, there was a statistically insig-

nificant difference between 2-year OS

rate of different HER2/neu protein score

categories. The latter results could be

explained by the relatively short duration

of follow-up and small number of pa-

tients in each HER2/neu protein score

category. In addition, Knoop et al.

(2005), had reported that recurrence is a

more precise measurement to outcome

than death as patients often have re-

ceived other treatment after their firstrelapse. Therefore, the effect of treat-

ment would confound the true predictive

impact of the tested biomarker. Studies

reported the possible relation between

HER2/neu protein and the outcome ofanthracycline-based chemotherapy

(Tubbs et al., 2009 & Konecny et al.,

2010). Additionally, HER2/neu-positive

tumors have been shown to have higherproliferation rate which might make

these tumors more sensitive to chemo-

therapy (Paik et al., 2008). On the other

hand, other studies showed conflicting

results which led to the suggestion that

HER2/neu could be a surrogate marker

for the true predictor(s) of anthracycline

sensitivity (Bozzetti et al., 2006 &

Munro et al., 2010). The current results,

although statistically insignificant, sug-

gested that 30.8% of HER2/neu negative

patients had better outcome (no relapse)after FAC therapy, making it difficult to

exclude FAC therapy in HER2/neu nega-

tive patients. This result is in agreement

to published reports (Esteva and Horto-

bagyi, 2009 and Miyoshi et al., 2010).

Furthermore, Miyoshi et al. (2010) ex-plained such observation through a sub-

set of HER2/neu-negative tumors (triple-

negative and basal-like breast cancer

subtypes) that were proposed to possess

a specific molecular phenotype. Such

phenotypes might partially explain the

sensitivity of some HER2/neu-negative

tumors to anthracycline treatment. In

addition, there might be an interplay of

other factors which gives further support

to the use of a panel of predictive mark-ers, rather than a single one, for tailoring

of chemotherapy. Among the relapsed

patients, HER2/neu failed to show a sig-

nificant relation to the type of relapse.

HER2/neu had no significant effect on

the time of relapse as well. In contrary tothese results, Freudenberg et al. (2009)

reported the possible role of HER2/neu

in regulating different aspects of tumor

growth and progression possibly through

increasing motility of tumor cells, de-

creasing apoptosis, enhancing signaling

interactions with the tumor microenvi-


Egypt. J. Med. Sci. 32 (1) 2011


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ronment, regulating adhesion, as well as

a multitude of other functions. The cur-rent results could be explained by the

limited number of the relapsed cases

and/or FAC therapy which might have

muffled the possible relation between

HER2/neu and each of time and type of

relapse.

In conclusion, HER2/neu protein

mightnt be considered as a predictor for

the outcome of FAC adjuvant chemo-therapy in Egyptian high risk female

breast cancer patients. Consequently,

large-scale prospective studies will beneeded to clearly define its predictive

value.

ACKNOWLEDGMENT

Special thanks go to Dr. Nadia

Abaas, Lecturer of Pathology, Faculty of

Medicine, University of Alexandria, for

her assistance in IHC work. We also

wish to thank Mrs. Safeya Abd Al-

Khalek, Pathology technician, for techni-cal assistance.

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