ORIGINAL ARTICLE

ErbB family immunohistochemical expression in colorectalcancer patients with higher risk of recurrence afterradical surgery

Glauco Baiocchi & Ademar Lopes & Renata A. Coudry & Benedito M. Rossi &Fernando A. Soares & Samuel Aguiar & Gustavo C. Guimarães & Fabio O. Ferreira &

Wilson T. Nakagawa

Accepted: 1 April 2009 /Published online: 24 April 2009# Springer-Verlag 2009

AbstractPurpose Investigate ErbB family expression in colorectalcancer patients with higher risk of recurrence after surgicaltreatment.Methods We studied 109 individuals with high risk stage IIand stage III patients submitted to radical surgery. ErbBexpression was assessed by tissue microarray technique.Results The immunohistochemical expression was consid-ered positive for EGFR, ErbB2, ErbB3, ErbB4 membrane,and ErbB4 cytoplasmic in respectively 57.8%, 8.3%,69.7%, 11%, and 19.3% of patients. ErbB3 negativeexpression was associated with lymphovascular invasion.EGFR, ErbB2, and cytoplasmic ErbB4 expression was notassociated with prognosis. Membranous positive ErbB4expression was an independent prognostic factor forrecurrence. ErbB3 negative expression was an independentprognostic factor for recurrence and survival in themultivariate analysis.

Conclusions The immunohistochemical expression ofErbB3 and ErbB4 may identify a subgroup with stage IIand III colorectal cancer at higher risk of recurrence.

Keywords Colonic neoplasm . Rectal neoplasm .

Prognosis . ErbB .Microarray analysis .

Immunohistochemistry

Introduction

Colorectal cancer (CRC) is one of the most common typesof cancer in the world and is the third cause of death in theUnited States and other Western countries [1]. Treatment ofCRC is based on curative surgery and followed by adjuvantchemotherapy when adverse prognostic factors are found.Patients with low rectal advanced tumors are bettersubmitted to neoadjuvant radiotherapy and chemotherapybefore surgery [2]. Until now, the best known prognosticfactor is the nodal status [3–5]. The use of adjuvantchemotherapy is recommended in cases which lymph nodesare affected (stage III) [6, 7] and there are also subpopu-lations of patients medically fit as stage II disease that couldbe considered for adjuvant therapy. This includes patientswith inadequately sampled lymph nodes, T4 lesions,perforation of the colon at tumor site, lymphovascularinvolvement, and poor differentiated histology [8].

In spite of multidisciplinary treatment, survival after5 years in these subgroups is under 60% and 70%. Somepatients probably have recurrence due to microscopicresidual disease resistant to the adjuvant treatment received.However, other patients do not have recurrent disease evenwithout adjuvant treatment as they have already been curedby surgery alone. Thus, there is a need to identify biological

Int J Colorectal Dis (2009) 24:1059–1068DOI 10.1007/s00384-009-0702-6

G. BaiocchiDepartment of Gynecologic Oncology,Hospital do Cancer AC Camargo,Sao Paulo, Brazil

A. Lopes :B. M. Rossi : S. Aguiar :G. C. Guimarães :F. O. Ferreira :W. T. NakagawaDepartment of Pelvic Surgery, Hospital do Cancer AC Camargo,Sao Paulo, Brazil

R. A. Coudry : F. A. SoaresDepartment of Pathology, Hospital do Cancer AC Camargo,Sao Paulo, Brazil

G. Baiocchi (*)Departamento de Ginecologia, Hospital do Câncer AC Camargo,Rua Antonio Prudente, 211,01509-010 Sao Paulo, Brazile-mail: glbaiocchi@yahoo.com.br

tumoral characteristics that may predict poor outcome andguide the development of new adjuvant treatments.

The epidermal growth factor receptor (EGFR/ErbB1),ErbB2/HER2, ErbB3/HER3, and ErbB4/HER4 are a groupof subtype I tyrosine kinases sharing structural homologies,especially at the intracellular domain. Protein kinases areenzymes that play a key regulation role in nearly everyaspect of cell biology [9–11]. They regulate apoptosis, cellcycle progression, cytoskeleton rearrangement, differentia-tion, development, and transcription. The basic structure ofErbB proteins consists of an extracellular domain, atransmembrane domain, and an intracellular domain. Afundamental aspect of signaling is the interaction of tworeceptors. The dimerization of two ErbB family membersand the transphosphorylation of their intracellular domaingenerate the initial signal leading to activation of numerousdownstream signaling pathways. The dimerization event isregulated by extracellular ligands of the epidermal growthfactor and neuregulin families [12, 13].

ErbB2 and ErbB3 are functionally incomplete trans-membrane receptors. They need interdependency. TheErbB2 extracellular domain is unique. It is locked in aconformation resembling the ligand-bound states of theother ErbB extracellular domains. ErbB2 is not regulated byligands as it has no ligands. ErbB2 is always available fordimerization with activated ErbB members. The ErbB3intracellular kinase domain is also unique. It is inactivecatalytically. However, ErbB3 is an efficient dimerizationpartner for all other ErbB members [14].

The signaling potency and selectivity of these pathwaysare proscribed by the dimer pair. There is a hierarchicalrelationship between the signaling potency and the ErbBdimers. Heterodimers are more active than homodimers,ErbB2 containing heterodimers are particularly active, andthe ErbB2–ErbB3 heterodimer is the most active dimer [15].

ErbB family signaling is deregulated in several subtypesof human cancers. This has been recognized most commonlyin breast cancer, lung cancer, and glioblastomas. EGFR isamplified and overexpressed in nearly half of all glioblasto-mas and is altered in kinase domain in 10% to 50% of non-small cell lung cancers [15]. ErbB2 protein overproductiondue to gene amplification is found in 25% to 30% of breastcancers and is related to worst prognosis [16, 17].

ErbB3 is not characterized currently as a proto-oncogeneand significant alterations have not been found in tumors.However, a significant data suggests that ErbB2 and ErbB3are partners in signaling [18, 19]. ErbB4 is the least well-characterized member of ErbB family. There is someevidence that does not implicate ErbB4 overactivity intumorigenesis and in fact it has been associated withdifferentiation and cell death [20].

There have been only a few studies that explored theexpression of ErbB family in CRC. The present study was

designed to investigate the immunohistochemical expres-sion of ErbB family in high risk CRC (high risk stage II andstage III) submitted to radical surgery and their role as aprognostic factor for recurrence and survival.

Materials and methods

Patients’ characteristics

A retrospective analysis was carried out in a series of 109individuals admitted at the A.C. Camargo Cancer Hospitalfrom January 1990 to August 2004. Patients who receivedprevious treatment at other institution and patients whoreceived chemotherapy or radiation therapy before surgerywere excluded. We also excluded patients that had tumorslocated at low and medium rectum. All patients had stageIII or stage II tumors with one or more bad prognosticfactors considered as: T4, presence of lymphatic, venous, orperineural invasion, high degree of differentiation, orpreoperative carcinoembryonic antigen (CEA) >10 ng/ml[21, 22]. Seventy seven (70.6%) patients had colon cancerand 32 (29.4%) had high rectal cancer. All were submittedto radical surgery (R0 resections).

The clinical and some pathological information werederived from medical records. The patients were stagedusing the TNM system [6]. There were 13 (11.9%) patientswith stage IIA, 33 (30.3%) stage IIB, seven (6.4%) stageIIIA, 35 (32.1%) stage IIIB, and 21 (19.3%) stage IIIC.

The prognostic factors used to consider the stage IIApatients as higher risk for recurrence were: three patientshad only CEA >10 ng/ml, four patients had tumors withonly perineural invasion, one patient had tumor with onlylymphatic invasion, one patient had tumor with onlyperineural invasion, one patient had tumor with lymphaticand perineural invasion, one patient had tumor withlymphatic and vessel invasion, and two patients had tumorswith vessel and perineural invasion.

Regarding adjuvant treatment, 19 (41.3%) stage IIpatients and 46 (73%) stage III patients were submitted tochemotherapy based on 5-fluouracil (5-FU) and leucovorin.At our institution, the routine use of adjuvant 5-FU-basedchemotherapy began in 1994.

The median follow-up time was 57.4 months (2–165.8).Only three patients missed their follow-up. The preopera-tive CEA was higher than 10 ng/ml in 24 patients and thevalue was missing in 37 (33.9%) patients.

Tissue microarray construction (TMA)

Hematoxylin- and eosin-stained (HE) sections of theprimary tumors were reviewed and areas of tumors weremarked on the slides. From each paraffin block, four tissue

1060 Int J Colorectal Dis (2009) 24:1059–1068

cores (0.6 mm in diameter) were sampled from eachmarked area in the donor block and mounted into arecipient paraffin block by a custom-made instrument(Beecher Instruments, Silver Springs, MD, USA). In theensuing paraffin array block, the tissue cylinders werealigned and marked for identification to a chart. Cores werespaced at intervals of 1 mm.

Immunohistochemical staining

TMA 3-μm sections were transferred to an adhesive-coatedslide system (Instrumentics Inc., Hackensack, NJ, USA) andthe antigen detection was carried out by the streptavidine–biotin–peroxidase technique (StreptABC, Dako®). Briefly,the slides were first coated with a silano solution (APTS-Sigma® A3648) diluted to 4% in acetone. The formalin-fixed, paraffin-embedded tissues were deparaffinized andprepared by successive passages through xylol and ethanoland submitted to antigenic recovery by pressure cooker heat,using a citrate buffer. Once the cuts were prepared, blockingof the peroxidase endogen with a 3% solution of hydrogenperoxide in methanol was carried out, followed by overnightincubation of the antibody. The reactions were alwaysaccompanied by positive control in tissue known to bepositive for the tested antibody and two negative controls.The first of these was carried out by the non-use of primaryantibody and the second by the removal of the secondaryantibody during the steps of the reaction. All the slides wereread by a single pathologist using a light microscopy, whowas intentionally not informed of clinical data.

The primary antibody used for EGFR was from Novocas-tra Laboratories Ltd., New Castle, UK (clone EGFR 113–mouse monoclonal antibody NCL-EGFR). It produced agranular or diffuse cytoplasmic staining. The primaryantibody used for ErbB2 was from Dako Corporation,Carpinteria, USA (anti-human c-erbB-2 oncoprotein– poly-clonal rabbit #A0485). It produced a membranous staining.The primary antibody used for ErbB3 was from Lab Vision,Fremont, USA (epitope specific rabbit antibody #RB-9211).It produced a granular or diffuse cytoplasmic staining. Theprimary antibody used for ErbB4 was from Lab Vision(#RB-9045). It produced a granular or diffuse cytoplasmicstaining and/or a membranous staining (Figs. 1, 2, 3, and 4).

From 109 primary patient tumors, 93 patients had fourtumor tissue cores analyzed, 11 patients had three tumortissue cores, and five patients had two tumor tissue cores.

Scoring system

Both membranous and cytoplasmic immunostaining wasevaluated semiquantitatively.

The cytoplasmic staining was scored as “0” when nostaining or weak staining in less than 10% of tumor cells;

“1+”, weak immunostaining in more than 10% of tumorcells; “2+”, moderate immunostaining in more than 10% oftumor cells; and “3+”, strong immunostaining in more than10% of tumor cells [23].

The membranous staining was scored as “0” when therewas no staining at all or membrane staining less than 10%of tumor cells; “1+”, defined as faint/barely perceptiblemembrane staining in more than 10% tumor cells; “2+”,defined as weak-to-moderate staining of the entire mem-brane in more than 10% tumor cells; “3+”, defined asstrong staining of the entire membrane in more than 10%tumor cells [24].

The values obtained from each core were added togetherand divided by the numbers of tumor cores evaluated. Acore tissue sample mean value was obtained. The meanvalues between 0 and 0.25 were arbitrarily considered as“negative”. The mean values between 0.26 and 1.4 were

Fig. 1 Microphotograph of EGFR immunohistochemical stainingwith “2+” cytoplasmic expression

Fig. 2 Microphotograph of ErbB2 immunohistochemical stainingwith “3+” membranous expression

Int J Colorectal Dis (2009) 24:1059–1068 1061

considered as “weak” immunostaining. The mean valuesbetween 1.5 and 2.4 were considered as “moderate”immunostaining. The mean values between 2.5 and 3 wereconsidered as “strong” immunostaining.

Finally, the immunostaining obtained were divided intotwo categories. “Negative” and “weak” immunostainingwere categorized as “Negative Expression” (mean from 0 to1.4). “Moderate” immunostaining and “strong” immunos-taining were categorized as “Positive Expression” (meanfrom 1.5 to 3).

Statistical analysis

The database was set up in the program “Statistical Packagefor Social Sciences” version 15.0 (SPSS Inc., Chicago, IL,USA). Follow-up time was considered to be from the dateof surgery up to the date of last information. The

association between immunostaining expression and othervariables was assessed by the chi-square test. The survivalcurves were estimated by the Kaplan–Meier method and thecomparison between the curves was made by the log-ranktest. The multivariate analysis was made by Cox regression.For all the tests made, an alpha error up to 5% (p<0.05)was considered.

Results

The demographic and tumor characteristics of the 109patients are summarized in Table 1. Thirty seven patientswere males (33.9%) and 72 were females (66.1%). Themedian age of the patients was 65 years (29–88 years).During the follow-up, 32 patients (29.3%) relapsed (sevenFig. 3 Microphotograph of ErbB3 immunohistochemical staining

with “1+” cytoplasmic expression

Fig. 4 Microphotograph of ErbB4 immunohistochemical stainingwith “3+” membranous and cytoplasmic expression

Table 1 Clinico-pathological characteristics of the 109 colorectalcancer patients

Variable No. of patients (%)

Age

<65 years 53 (48.6)

>65 years 56 (51.4)

Gender

Male 37 (33.9)

Female 72 (66.1)

Location

Colon 77 (70.6)

Rectum 32 (29.4)

Adjuvant chemotherapy

No 44 (40.4)

Yes 65 (59.6)

Lymphatic invasion

Absent 79 (72.5)

Present 30 (27.5)

Venous invasion

Absent 93 (85.3)

Present 16 (14.7)

Perineural invasion

Absent 86 (78.9)

Present 23 (21.1)

Histological grade

Low 27 (25.2)

High 80 (74.8)

Stage

IIA 13 (11.9)

IIB 33 (30.3)

IIIA 7 (6.4)

IIIB 35 (32.1)

IIIC 21 (19.3)

1062 Int J Colorectal Dis (2009) 24:1059–1068

local recurrence and 25 distant metastasis) and 27 (24.8%)died from disease. The 5-year overall survival was 69.7%and 5-year disease-free survival was 66.1%.

Immunohistochemical expression status

Forty six patients (42.2%) had tumors with negativeexpression of EGFR and 63 (57.8%) had positive expres-sion. One hundred (91.7%) patients had tumors withnegative expression of ErbB2 and only nine patients hadpositive expression (8.3%). ErbB3 had negative expressionin 33 (30.3%) patients and positive expression in 76(69.7%) patients. Regarding ErbB4, 97 (89%) had negativemembranous expression whereas 88 (80.7%) had negativecytoplasmic expression (Table 2).

Association tests among variables

Regarding ErbB3 expression, it was positive in 78.5% oftumors with absence of lymphovascular invasion and in46.7% of tumors with presence of lymphovascular invasion(p=0.001).

There was no statistical difference in the distribution ofEGFR, ErbB2, and ErbB4 expression among the variables.

Five-year recurrence

Analyzing the total sample, presence of lymphatic invasion(p=0.008), presence of perineural invasion (p=0.048),negative expression of ErbB3 (p=0.012) (Fig. 5), andpositive membranous expression of ErbB4 (p=0.022)(Fig. 6) were the only variables that influenced the risk ofrecurrence in univariate analysis (Tables 3 and 4). Regard-ing multivariate analysis, negative expression of ErbB3(HR 2.19; 95% CI=1.11–4.35) and positive membranousexpression of ErbB4 (HR 2.73; 95% CI=1.17–6.37)maintained as an independent risk of recurrence even whenadjusted to lymphatic invasion and perineural invasion inthe Cox regression model (Table 5).

Five-year overall survival

Analyzing the total sample, presence of lymphatic inva-sion (p=0.009) and negative expression of ErbB3 (p=0.004) (Fig. 7) were the only variables that influenced therisk of death in univariate analysis (Tables 6 and 7).

Table 2 ErbB family expression for the 109 colorectal cancer patients

Variable Expression No. of patients (%)

EGFR Negative 46 (42.2)

Positive 63 (57.8)

ErbB2 Negative 100 (91.7)

Positive 9 (8.3)

ErbB3 Negative 33 (30.3)

Positive 76 (69.7)

ErbB4 (membrane) Negative 97 (89.0)

Positive 12 (11.0)

ErbB4 (cytoplasmic) Negative 88 (80.7)

Positive 21 (19.3)

Negative expression

Positive expression

D

isea

se f

ree

surv

ival

(%

)

p=0.012

months

Fig. 5 Disease-free survivalKaplan–Meier curves fornegative and positive expressionof ErbB3 (p=0.012)

Int J Colorectal Dis (2009) 24:1059–1068 1063

Regarding multivariate analysis, only negative expressionof ErbB3 (HR 2.46; 95% CI=1.19–5.08) maintained as anindependent risk of recurrence even when adjusted tolymphatic invasion, perineural invasion and positivemembranous expression of ErbB4 in the Cox regressionmodel (Table 8).

Discussion

It is estimated that less than one third of the patients newlydiagnosed with colorectal cancer will have node positivedisease (stage III) and about one quarter will have nodenegative disease (stage II) [8].

Although patients with stage II colon cancer aregenerally considered to have a good prognosis after surgeryalone, approximately one quarter will experience recurrencewithin 5 years [8]. Although there are markers for high riskstage II colon cancer, it should be cautioned that theidentification of such markers may simply indicate a patientat higher risk for recurrence or death, without necessarilyleading to the conclusion that adjuvant therapy will be ofsignificant clinical benefit.

Despite the significant improvement in traditional andnew chemotherapy regimens, the main efforts of recent

Table 3 Univariate analysis related to clinico-pathological character-istics and disease-free survival

Variable 5-year survival (%) p value

Localization

Colon 68.8 0.48Rectum 59.4

Stage

II 71.7 0.30III 61.9

Venous invasion

Absent 68.8 0.11Present 50.0

Lymphatic invasion

Absent 73.4 0.008Present 46.7

Perineural invasion

Absent 70.9 0.048Present 47.8

Histological grade

Low 59.3 0.34High 67.5

Adjuvant chemotherapy

No 65.9 0.84Yes 66.2

Table 4 Univariate analysis related to EGFR, ErbB2, ErbB3, andErbB4 expression and disease-free survival

Variable Expression 5-year survival (%) p value

EGFR Negative 69.6 0.41Positive 63.5

ErbB2 Negative 66.0 0.89Positive 66.7

ErbB3 Negative 51.5 0.012Positive 72.4

ErbB4 (membrane) Negative 69.1 0.022Positive 41.7

ErbB4 (cytoplasmic) Negative 65.9 0.97Positive 66.7

Negative expression

Positive expression

Dis

ease

fre

e su

rviv

al (

%)

p=0.022

months

Fig. 6 Disease-free survivalKaplan–Meier curves for nega-tive and positive ErbB4membranous expression(p=0.022)

1064 Int J Colorectal Dis (2009) 24:1059–1068

researches are focused on the potential use of targetedtherapy.

Type I tyrosine kinase receptors and their signal trans-duction pathways have a crucial role in cancer biology, butconflicting data still exist regarding the immunohistochem-ical expression of EGFR, ErbB2, ErbB3, and ErbB4 incolorectal cancer and its role in risk of recurrence and death.

Some studies investigated the immunohistochemicalexpression of EGFR in CRC. The incidence of EGFRexpression ranges from 26.6% to 80% [25, 26]. Yasui et al.[25] first described EGFR expression in 26.6% in stage IIand III colon cancers. McKay et al. [27] evaluated EGFRexpression in 249 colorectal tumors and its correlation withlymph node metastasis. They found positive expression in72.7% of primary tumors (29.3% only membranous expres-sion, 37.8% membranous and cytoplasmic expression, and5.6% only cytoplasmic expression) and there was noinfluence in survival. Only 40.5% samples showed equiva-lent expression in paired colorectal tumors and lymph node

metastasis. Scartozzi et al. [28] also did not find correlationwith EGFR expression in primary colorectal tumors and theirmetastatic site (hepatic, pulmonary, central nervous system,and bone metastasis). EGFR was evaluated in 99 patientsand considered positive in 53% primary tumors. In 36% ofprimary tumors expressing EGFR, the corresponding meta-static site was found negative, whereas it was found positivein 15% of patients from EGFR negative primary cancers.

Table 5 Association between ErbB3 expression, ErbB4 membranousexpression, and the risk of recurrence (multivariate analysis)

Variable HR 95% CI p value

Presence of lymphatic invasion 1.77 0.90–3.49 0.98

ErbB3 negative expression 2.19 1.11–4.35 0.020

ErbB4 positive membranousexpression

2.73 1.17–6.37 0.024

Presence of perineural invasion 1.79 0.87–3.70 0.11

Estimated risk from Cox regression model with matching variableslymphovascular invasion, ErbB3 expression, ErbB4 expression, andperineural invasion

HR hazard ratio, CI confidence interval

Negative expression

Positive expression

Ove

rall

surv

ival

(%

)

p=0.004

months

Fig. 7 Overall survival Kaplan–Meier curves for negativeand positive expression ofErbB3 (p=0.004)

Table 6 Univariate analysis related to clinico-pathological character-istics and overall survival

Variable 5-year survival (%) p value

Localization

Colon 70.1 0.94Rectum 68.8

Stage

II 71.7 0.34III 8.3

Venous invasion

Absent 71.0 0.51Present 62.5

Lymphatic invasion

Absent 77.2 0.009Present 50.0

Perineural invasion

Absent 73.3 0.15Present 56.5

Histological grade

Low 66.7 0.68High 71.3

Adjuvant chemotherapy

No 59.1 0.15Yes 76.9

Int J Colorectal Dis (2009) 24:1059–1068 1065

Goldstein and Armin [29] reviewed 102 patients withcolorectal cancer and observed EGFR expression in 75.5%of cases. They analyzed the hypothesis that EGFR expres-sion may change during the process of cancer invasion andfound correlation with the risk of recurrence and death whenEGFR was expressed in the deep regions of colorectalcancers. Other authors investigated the prognostic signifi-cance of EGFR expression in colorectal cancers. Spano et al.[26] examined tumor samples from 150 patients and foundEGFR overexpression in 80% of cases and it was associatedwith TNM stage T3, but with no survival correlation. In theother hand, Steele et al. [30] observed in 50 patientssignificant EGFR expression in stage III and poor differen-tiated tumors, with poor correlation to survival. This fact wasalso seen in Galizia et al. [31] study of 126 colon cancerpatients, submitted to curative surgery. EGFR expressionwere detected in 35.6% of patients and was correlated withdisease recurrence and worse survival in both univariate andmultivariate analysis.

In the current study, positive expression of EGFR wasfound in 57.8% of patients and, as in most of the series,there was no correlation with recurrence and overallsurvival.

The ErbB2 expression was evaluated by some series andthe majority could not find any correlation with itsexpression and prognosis. Nathanson et al. [32] showedcorrelation between gene amplification and immunohisto-chemical expression of ErbB2 in 139 patients with coloncancer. Only 3.6% of tumors expressed ErbB2 and therewas no association either with stage or with survival.Schuell et al. [33] and Ochs et al. [34] also foundmembranous expression in a few primary colorectal tumors,respectively 30% and 11% of 77 and 109 patients. McKayet al. [35] evaluated ErbB2 expression in 249 primarycolorectal tumors and 42 lymph node metastasis. Theyfound positive expression in 81.8% of patients withconcordant staining with lymph node metastasis in only52.4% of cases. Colon cancers were more likely to expressErbB2 than rectal cancers. They also showed no associationwith gene polymorphism and no correlation with survival.The cytoplasmic expression of ErbB2 was evaluated by twoseries, with conflicting results. Essapen et al. [36] noted in170 colorectal tumors membranous expression in 41% andcytoplasmic expression in 87% of the patients. Thecytoplasmic expression was related to better prognosis instage III patients. However, Osako et al. [37] reported in146 colorectal tumors membranous expression in 3% andcytoplasmic expression in 68.5% of the cases. Thecytoplasmic expression had correlation with worse overallsurvival in stage II patients.

In the current study, we found ErbB2 membranouspositive expression in only 8.3% of cases. As in EGFRexpression, our series found no association between thepositive expression of ErbB2 and the increase risk ofrecurrence and death. Other studies evaluated the co-expression of EGFR and ErbB2. Kluftinger et al. [38]showed co-expression in 17% of 35 cases and Cunninghamet al. [39] in 75% of 87 patients. The latter found EGFR andErbB2 cytoplasmic expression in 63% and 75% of cases. Inour study, we found co-expression of EGFR and ErbB2 inonly 7.9% of the patients.

In CRC, only a few studies investigated the proteinexpression of ErbB3 and ErbB4. Maurer et al. [40] revealedan ErbB3 expression rate of 89% in colorectal tumors bothmembranous and cytoplasmic, with co-expression withErbB2 in 77% of the cases. Kapitanovic et al. [41] dem-onstrated that ErbB3 is expressed in 78% of cases, and wasexclusively cytoplasmic. Until now, Lee at al. [42] publishedthe only article that had examined the expression of all ErbBfamily proteins in patients with colorectal cancer. Onlymembranous staining was considered positive. They evalu-ated 125 patients submitted to curative surgery and consid-ered EGFR, ErbB2, ErbB3, and ErbB4 positive expressionrespectively in 52%, 35%, 36%, and 22% of cases. Asignificantly higher percentage of overexpressed ErbB3 wasobserved in early stage carcinomas (stages I and II).

Table 7 Univariate analysis related to EGFR, ErbB2, ErbB3, andErbB4 expression and overall survival

Variable Expression 5-year survival (%) p value

EGFR Negative 76.1 0.24Positive 65.1

ErbB2 Negative 71.0 0.36Positive 55.6

ErbB3 Negative 51.5 0.004Positive 77.6

ErbB4 (membrane) Negative 70.1 0.31Positive 66.7

ErbB4 (cytoplasmic) Negative 68.2 0.55Positive 76.2

Table 8 Association between ErbB3 expression and the risk of death(multivariate analysis)

Variable HR 95% CI p value

Presence of lymphatic invasion 1.81 0.89–3.68 0.10

ErbB3 negative expression 2.46 1.19–5.08 0.014

ErbB4 positive membranousexpression

1.79 0.60–5.33 0.29

Presence of perineural invasion 1.69 0.77–3.70 0.18

Estimated risk from Cox regression model with matching variableslymphovascular invasion, ErbB3 expression, ErbB4 expression, andperineural invasion

HR hazard ratio, CI confidence interval

1066 Int J Colorectal Dis (2009) 24:1059–1068

However, ErbB4 was more overexpressed in advanced stagecancers (stages III and IV). The expression of EGFR, ErbB2,ErbB3, and ErbB4 alone denoted no association with ashortened survival, but patients with simultaneous over-expression of ErbB2 and ErbB4 had a shorter overallsurvival time in only the univariate analysis, probablybecause of correlation with more advanced stages. Koun-tourakis et al. [43] evaluated the ErbB3 and ErbB4expression in 106 colorectal tumors. Both membranous andcytoplasmic expression was identified. The rates of expres-sion for ErbB3 and ErbB4 respectively were 17% and 18.9%membranous; 28.3% and 30.2% cytoplasmic. ErbB3 positivecytoplasmic expression was associated with moderate tumorgrade and ErbB4 membranous expression was associatedwith lymph node involvement. There was no correlationbetween the ErbB3 and ErbB4 expression and patientprognosis.

In our series, negative expression of ErbB3 was signifi-cantly associated with the presence of lymphovascularinvasion. However, ErbB3 negative expression maintainedin multivariate analysis, the negative impact both in risk ofrecurrence and death.

ErbB3 protein has low affinity to ligands when comparedto other receptors of ErbB family. The low expression mayindirectly increase or perpetuate other signaling pathways.Another hypothesis is the possibility that the cytoplasmicsequestering of ErbB3 receptors may alter the propensity toform ErbB3-containing heterodimers at the level of plasmamembrane [44]. There is large evidence that ligand-inducedheterodimerization of ErbB family receptors is necessary todiversify and specify biological responses. The role of ErbB3protein to act as an efficient suppressor of ligand-inducedErbB4 signaling was also described [45] and the over-expression of ErbB3 somehow may inhibit signalingtranscription induced by ErbB family receptors ligands.

As previously mentioned, positive membranous expres-sion of ErbB4 influenced the risk of recurrence both inunivariate and multivariate analysis. There was no correla-tion to overall survival. The cytoplasmic expression ofErbB4 had no prognostic correlation to relapse and death.

There are several possible reasons for discrepanciesbetween studies. The small sample size, the disparatescoring systems used to classify family ErbB overexpres-sion, the source of antibody used, and the immunohisto-chemical protocol make comparison between the studiesvery challenging.

Scoring systems for tumor markers in CRC are usuallybased on a measure of proportion of positive tumor cellsand combined with a degree of staining intensity [46]. It isrecognized that the interpretation of staining intensity is notonly highly subjective but may be affected by storage time,variation in protocols, and fixation procedures. Despitethese concerns, staining intensity has become an integral

component of many immunohistochemical scoring methodsfor tumor markers in colorectal cancer.

Goethals et al. [47] has reported that four cores biopsiesare sufficient to account for tumor heterogeneity. Although itis argued that a single core sample (0.6 mm) per tumor maynot be representative of the whole tumor, results even usingone sample tumor have shown well-established associationsbetween molecular features and clinico-pathological end-points [48, 49]. TMA technology allowed us to analyze 415tumor samples. Ninety three patients had four tumorsamples, 11 patients had three tumor samples, and fivepatients had two tumor samples. We had no primary tumorsampled with one core.

As far as we know, the current series is the first to suggest aprognostic correlation between ErbB3 and ErbB4 immunos-taining expression in only stage II and III colorectal primarytumors after curative surgery using the tissue microarraytechnique. Our study is easily reproducible and suggests apossible role of ErbB3 and ErbB4 in CRC carcinogenesis.More complete knowledge about prognostic and predictivefactors will allow clinicians to identify those patients at higherrisk of recurrence who are more likely to benefit from newadjuvant treatments.

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