ErbB family immunohistochemical expression in colorectal cancer patients with higher risk of recurrence after radical surgery

Int J Colorectal Dis (2009) 24:1059–1068 DOI 10.1007/s00384-009-0702-6 ORIGINAL ARTICLE ErbB family immunohistochemical expression in colorectal cancer patients with higher risk of recurrence after radical 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 Abstract Purpose Investigate ErbB family expression in colorectal cancer patients with higher risk of recurrence after surgical treatment. Methods We studied 109 individuals with high risk stage II and stage III patients submitted to radical surgery. ErbB expression was assessed by tissue microarray technique. Results The immunohistochemical expression was considered 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 negative expression was associated with lymphovascular invasion. EGFR, ErbB2, and cytoplasmic ErbB4 expression was not associated with prognosis. Membranous positive ErbB4 expression was an independent prognostic factor for recurrence. ErbB3 negative expression was an independent prognostic factor for recurrence and survival in the multivariate analysis. G. Baiocchi Department 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. Nakagawa Department of Pelvic Surgery, Hospital do Cancer AC Camargo, Sao Paulo, Brazil R. A. Coudry : F. A. Soares Department 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, Brazil e-mail: glbaiocchi@yahoo.com.br Conclusions The immunohistochemical expression of ErbB3 and ErbB4 may identify a subgroup with stage II and 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 types of cancer in the world and is the third cause of death in the United States and other Western countries [1]. Treatment of CRC is based on curative surgery and followed by adjuvant chemotherapy when adverse prognostic factors are found. Patients with low rectal advanced tumors are better submitted to neoadjuvant radiotherapy and chemotherapy before surgery [2]. Until now, the best known prognostic factor is the nodal status [3–5]. The use of adjuvant chemotherapy is recommended in cases which lymph nodes are affected (stage III) [6, 7] and there are also subpopulations of patients medically fit as stage II disease that could be considered for adjuvant therapy. This includes patients with inadequately sampled lymph nodes, T4 lesions, perforation of the colon at tumor site, lymphovascular involvement, and poor differentiated histology [8]. In spite of multidisciplinary treatment, survival after 5 years in these subgroups is under 60% and 70%. Some patients probably have recurrence due to microscopic residual disease resistant to the adjuvant treatment received. However, other patients do not have recurrent disease even without adjuvant treatment as they have already been cured by surgery alone. Thus, there is a need to identify biological 1060 tumoral characteristics that may predict poor outcome and guide the development of new adjuvant treatments. The epidermal growth factor receptor (EGFR/ErbB1), ErbB2/HER2, ErbB3/HER3, and ErbB4/HER4 are a group of subtype I tyrosine kinases sharing structural homologies, especially at the intracellular domain. Protein kinases are enzymes that play a key regulation role in nearly every aspect of cell biology [9–11]. They regulate apoptosis, cell cycle progression, cytoskeleton rearrangement, differentiation, development, and transcription. The basic structure of ErbB proteins consists of an extracellular domain, a transmembrane domain, and an intracellular domain. A fundamental aspect of signaling is the interaction of two receptors. The dimerization of two ErbB family members and the transphosphorylation of their intracellular domain generate the initial signal leading to activation of numerous downstream signaling pathways. The dimerization event is regulated by extracellular ligands of the epidermal growth factor and neuregulin families [12, 13]. ErbB2 and ErbB3 are functionally incomplete transmembrane receptors. They need interdependency. The ErbB2 extracellular domain is unique. It is locked in a conformation resembling the ligand-bound states of the other ErbB extracellular domains. ErbB2 is not regulated by ligands as it has no ligands. ErbB2 is always available for dimerization with activated ErbB members. The ErbB3 intracellular kinase domain is also unique. It is inactive catalytically. However, ErbB3 is an efficient dimerization partner for all other ErbB members [14]. The signaling potency and selectivity of these pathways are proscribed by the dimer pair. There is a hierarchical relationship between the signaling potency and the ErbB dimers. Heterodimers are more active than homodimers, ErbB2 containing heterodimers are particularly active, and the ErbB2–ErbB3 heterodimer is the most active dimer [15]. ErbB family signaling is deregulated in several subtypes of human cancers. This has been recognized most commonly in breast cancer, lung cancer, and glioblastomas. EGFR is amplified and overexpressed in nearly half of all glioblastomas and is altered in kinase domain in 10% to 50% of nonsmall cell lung cancers [15]. ErbB2 protein overproduction due to gene amplification is found in 25% to 30% of breast cancers and is related to worst prognosis [16, 17]. ErbB3 is not characterized currently as a proto-oncogene and significant alterations have not been found in tumors. However, a significant data suggests that ErbB2 and ErbB3 are partners in signaling [18, 19]. ErbB4 is the least wellcharacterized member of ErbB family. There is some evidence that does not implicate ErbB4 overactivity in tumorigenesis and in fact it has been associated with differentiation and cell death [20]. There have been only a few studies that explored the expression of ErbB family in CRC. The present study was Int J Colorectal Dis (2009) 24:1059–1068 designed to investigate the immunohistochemical expression of ErbB family in high risk CRC (high risk stage II and stage III) submitted to radical surgery and their role as a prognostic factor for recurrence and survival. Materials and methods Patients’ characteristics A retrospective analysis was carried out in a series of 109 individuals admitted at the A.C. Camargo Cancer Hospital from January 1990 to August 2004. Patients who received previous treatment at other institution and patients who received chemotherapy or radiation therapy before surgery were excluded. We also excluded patients that had tumors located at low and medium rectum. All patients had stage III or stage II tumors with one or more bad prognostic factors considered as: T4, presence of lymphatic, venous, or perineural invasion, high degree of differentiation, or preoperative carcinoembryonic antigen (CEA) >10 ng/ml [21, 22]. Seventy seven (70.6%) patients had colon cancer and 32 (29.4%) had high rectal cancer. All were submitted to radical surgery (R0 resections). The clinical and some pathological information were derived from medical records. The patients were staged using the TNM system [6]. There were 13 (11.9%) patients with stage IIA, 33 (30.3%) stage IIB, seven (6.4%) stage IIIA, 35 (32.1%) stage IIIB, and 21 (19.3%) stage IIIC. The prognostic factors used to consider the stage IIA patients as higher risk for recurrence were: three patients had only CEA >10 ng/ml, four patients had tumors with only perineural invasion, one patient had tumor with only lymphatic invasion, one patient had tumor with only perineural invasion, one patient had tumor with lymphatic and perineural invasion, one patient had tumor with lymphatic and vessel invasion, and two patients had tumors with vessel and perineural invasion. Regarding adjuvant treatment, 19 (41.3%) stage II patients and 46 (73%) stage III patients were submitted to chemotherapy based on 5-fluouracil (5-FU) and leucovorin. At our institution, the routine use of adjuvant 5-FU-based chemotherapy began in 1994. The median follow-up time was 57.4 months (2–165.8). Only three patients missed their follow-up. The preoperative CEA was higher than 10 ng/ml in 24 patients and the value was missing in 37 (33.9%) patients. Tissue microarray construction (TMA) Hematoxylin- and eosin-stained (HE) sections of the primary tumors were reviewed and areas of tumors were marked on the slides. From each paraffin block, four tissue Int J Colorectal Dis (2009) 24:1059–1068 1061 cores (0.6 mm in diameter) were sampled from each marked area in the donor block and mounted into a recipient paraffin block by a custom-made instrument (Beecher Instruments, Silver Springs, MD, USA). In the ensuing paraffin array block, the tissue cylinders were aligned and marked for identification to a chart. Cores were spaced at intervals of 1 mm. Immunohistochemical staining TMA 3-μm sections were transferred to an adhesive-coated slide system (Instrumentics Inc., Hackensack, NJ, USA) and the antigen detection was carried out by the streptavidine– biotin–peroxidase technique (StreptABC, Dako®). Briefly, the slides were first coated with a silano solution (APTSSigma® A3648) diluted to 4% in acetone. The formalinfixed, paraffin-embedded tissues were deparaffinized and prepared by successive passages through xylol and ethanol and submitted to antigenic recovery by pressure cooker heat, using a citrate buffer. Once the cuts were prepared, blocking of the peroxidase endogen with a 3% solution of hydrogen peroxide in methanol was carried out, followed by overnight incubation of the antibody. The reactions were always accompanied by positive control in tissue known to be positive for the tested antibody and two negative controls. The first of these was carried out by the non-use of primary antibody and the second by the removal of the secondary antibody during the steps of the reaction. All the slides were read by a single pathologist using a light microscopy, who was intentionally not informed of clinical data. The primary antibody used for EGFR was from Novocastra Laboratories Ltd., New Castle, UK (clone EGFR 113– mouse monoclonal antibody NCL-EGFR). It produced a granular or diffuse cytoplasmic staining. The primary antibody used for ErbB2 was from Dako Corporation, Carpinteria, USA (anti-human c-erbB-2 oncoprotein– polyclonal 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. The primary antibody used for ErbB4 was from Lab Vision (#RB-9045). It produced a granular or diffuse cytoplasmic staining and/or a membranous staining (Figs. 1, 2, 3, and 4). From 109 primary patient tumors, 93 patients had four tumor tissue cores analyzed, 11 patients had three tumor tissue cores, and five patients had two tumor tissue cores. Fig. 1 Microphotograph of EGFR immunohistochemical staining with “2+” cytoplasmic expression “1+”, weak immunostaining in more than 10% of tumor cells; “2+”, moderate immunostaining in more than 10% of tumor cells; and “3+”, strong immunostaining in more than 10% of tumor cells [23]. The membranous staining was scored as “0” when there was no staining at all or membrane staining less than 10% of tumor cells; “1+”, defined as faint/barely perceptible membrane staining in more than 10% tumor cells; “2+”, defined as weak-to-moderate staining of the entire membrane in more than 10% tumor cells; “3+”, defined as strong staining of the entire membrane in more than 10% tumor cells [24]. The values obtained from each core were added together and divided by the numbers of tumor cores evaluated. A core tissue sample mean value was obtained. The mean values between 0 and 0.25 were arbitrarily considered as “negative”. The mean values between 0.26 and 1.4 were Scoring system Both membranous and cytoplasmic immunostaining was evaluated semiquantitatively. The cytoplasmic staining was scored as “0” when no staining or weak staining in less than 10% of tumor cells; Fig. 2 Microphotograph of ErbB2 immunohistochemical staining with “3+” membranous expression 1062 Int J Colorectal Dis (2009) 24:1059–1068 association between immunostaining expression and other variables was assessed by the chi-square test. The survival curves were estimated by the Kaplan–Meier method and the comparison between the curves was made by the log-rank test. 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 Fig. 3 Microphotograph of ErbB3 immunohistochemical staining with “1+” cytoplasmic expression considered as “weak” immunostaining. The mean values between 1.5 and 2.4 were considered as “moderate” immunostaining. The mean values between 2.5 and 3 were considered as “strong” immunostaining. Finally, the immunostaining obtained were divided into two categories. “Negative” and “weak” immunostaining were categorized as “Negative Expression” (mean from 0 to 1.4). “Moderate” immunostaining and “strong” immunostaining were categorized as “Positive Expression” (mean from 1.5 to 3). Statistical analysis The database was set up in the program “Statistical Package for Social Sciences” version 15.0 (SPSS Inc., Chicago, IL, USA). Follow-up time was considered to be from the date of surgery up to the date of last information. The Fig. 4 Microphotograph of ErbB4 immunohistochemical staining with “3+” membranous and cytoplasmic expression The demographic and tumor characteristics of the 109 patients are summarized in Table 1. Thirty seven patients were males (33.9%) and 72 were females (66.1%). The median age of the patients was 65 years (29–88 years). During the follow-up, 32 patients (29.3%) relapsed (seven Table 1 Clinico-pathological characteristics of the 109 colorectal cancer patients Variable Age <65 years >65 years Gender Male Female Location Colon Rectum Adjuvant chemotherapy No Yes Lymphatic invasion Absent Present Venous invasion Absent Present Perineural invasion Absent Present Histological grade Low High Stage IIA IIB IIIA IIIB IIIC No. of patients (%) 53 56 (48.6) (51.4) 37 72 (33.9) (66.1) 77 32 (70.6) (29.4) 44 65 (40.4) (59.6) 79 30 (72.5) (27.5) 93 (85.3) 16 (14.7) 86 23 (78.9) (21.1) 27 80 (25.2) (74.8) 13 33 7 35 21 (11.9) (30.3) (6.4) (32.1) (19.3) Int J Colorectal Dis (2009) 24:1059–1068 1063 Table 2 ErbB family expression for the 109 colorectal cancer patients Variable Expression No. of patients (%) EGFR Negative Positive Negative Positive Negative Positive Negative Positive 46 63 100 9 33 76 97 12 (42.2) (57.8) (91.7) (8.3) (30.3) (69.7) (89.0) (11.0) Negative Positive 88 21 (80.7) (19.3) ErbB2 ErbB3 ErbB4 (membrane) ErbB4 (cytoplasmic) 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 negative expression of EGFR and 63 (57.8%) had positive expression. One hundred (91.7%) patients had tumors with negative expression of ErbB2 and only nine patients had positive expression (8.3%). ErbB3 had negative expression in 33 (30.3%) patients and positive expression in 76 (69.7%) patients. Regarding ErbB4, 97 (89%) had negative membranous expression whereas 88 (80.7%) had negative cytoplasmic expression (Table 2). Association tests among variables Regarding ErbB3 expression, it was positive in 78.5% of tumors with absence of lymphovascular invasion and in 46.7% of tumors with presence of lymphovascular invasion (p=0.001). There was no statistical difference in the distribution of EGFR, 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), and positive membranous expression of ErbB4 (p= 0.022) (Fig. 6) were the only variables that influenced the risk of recurrence in univariate analysis (Tables 3 and 4). Regarding multivariate analysis, negative expression of ErbB3 (HR 2.19; 95% CI=1.11–4.35) and positive membranous expression of ErbB4 (HR 2.73; 95% CI = 1.17–6.37) maintained as an independent risk of recurrence even when adjusted to lymphatic invasion and perineural invasion in the Cox regression model (Table 5). Five-year overall survival Analyzing the total sample, presence of lymphatic invasion (p=0.009) and negative expression of ErbB3 (p= 0.004) (Fig. 7) were the only variables that influenced the risk of death in univariate analysis (Tables 6 and 7). Disease free survival (%) Fig. 5 Disease-free survival Kaplan–Meier curves for negative and positive expression of ErbB3 (p=0.012) Positive expression Negative expression p=0.012 months 1064 Int J Colorectal Dis (2009) 24:1059–1068 Fig. 6 Disease-free survival Kaplan–Meier curves for negative and positive ErbB4 membranous expression (p=0.022) Disease free survival (%) Negative expression Positive expression p=0.022 months Regarding multivariate analysis, only negative expression of ErbB3 (HR 2.46; 95% CI=1.19–5.08) maintained as an independent risk of recurrence even when adjusted to lymphatic invasion, perineural invasion and positive membranous expression of ErbB4 in the Cox regression model (Table 8). Table 3 Univariate analysis related to clinico-pathological characteristics and disease-free survival Variable Localization Colon Rectum Stage II III Venous invasion Absent Present Lymphatic invasion Absent Present Perineural invasion Absent Present Histological grade Low High Adjuvant chemotherapy No Yes 5-year survival (%) p value 68.8 59.4 0.48 71.7 61.9 0.30 68.8 50.0 0.11 73.4 46.7 0.008 70.9 47.8 0.048 59.3 67.5 0.34 65.9 66.2 0.84 Discussion It is estimated that less than one third of the patients newly diagnosed with colorectal cancer will have node positive disease (stage III) and about one quarter will have node negative disease (stage II) [8]. Although patients with stage II colon cancer are generally considered to have a good prognosis after surgery alone, approximately one quarter will experience recurrence within 5 years [8]. Although there are markers for high risk stage II colon cancer, it should be cautioned that the identification of such markers may simply indicate a patient at higher risk for recurrence or death, without necessarily leading to the conclusion that adjuvant therapy will be of significant clinical benefit. Despite the significant improvement in traditional and new chemotherapy regimens, the main efforts of recent Table 4 Univariate analysis related to EGFR, ErbB2, ErbB3, and ErbB4 expression and disease-free survival Variable Expression EGFR Negative Positive Negative Positive Negative Positive Negative Positive Negative Positive ErbB2 ErbB3 ErbB4 (membrane) ErbB4 (cytoplasmic) 5-year survival (%) p value 69.6 63.5 66.0 66.7 51.5 72.4 69.1 41.7 65.9 66.7 0.41 0.89 0.012 0.022 0.97 Int J Colorectal Dis (2009) 24:1059–1068 1065 Table 5 Association between ErbB3 expression, ErbB4 membranous expression, and the risk of recurrence (multivariate analysis) Table 6 Univariate analysis related to clinico-pathological characteristics and overall survival Variable HR 95% CI Variable Presence of lymphatic invasion ErbB3 negative expression ErbB4 positive membranous expression Presence of perineural invasion 1.77 2.19 2.73 0.90–3.49 1.11–4.35 1.17–6.37 0.98 0.020 0.024 1.79 0.87–3.70 0.11 p value Estimated risk from Cox regression model with matching variables lymphovascular invasion, ErbB3 expression, ErbB4 expression, and perineural invasion HR hazard ratio, CI confidence interval researches are focused on the potential use of targeted therapy. Type I tyrosine kinase receptors and their signal transduction pathways have a crucial role in cancer biology, but conflicting data still exist regarding the immunohistochemical expression of EGFR, ErbB2, ErbB3, and ErbB4 in colorectal cancer and its role in risk of recurrence and death. Some studies investigated the immunohistochemical expression of EGFR in CRC. The incidence of EGFR expression ranges from 26.6% to 80% [25, 26]. Yasui et al. [25] first described EGFR expression in 26.6% in stage II and III colon cancers. McKay et al. [27] evaluated EGFR expression in 249 colorectal tumors and its correlation with lymph node metastasis. They found positive expression in 72.7% of primary tumors (29.3% only membranous expression, 37.8% membranous and cytoplasmic expression, and 5.6% only cytoplasmic expression) and there was no influence in survival. Only 40.5% samples showed equivalent expression in paired colorectal tumors and lymph node Fig. 7 Overall survival Kaplan– Meier curves for negative and positive expression of ErbB3 (p=0.004) Localization Colon Rectum Stage II III Venous invasion Absent Present Lymphatic invasion Absent Present Perineural invasion Absent Present Histological grade Low High Adjuvant chemotherapy No Yes 5-year survival (%) p value 70.1 68.8 0.94 71.7 8.3 0.34 71.0 62.5 0.51 77.2 50.0 0.009 73.3 56.5 0.15 66.7 71.3 0.68 59.1 76.9 0.15 metastasis. Scartozzi et al. [28] also did not find correlation with EGFR expression in primary colorectal tumors and their metastatic site (hepatic, pulmonary, central nervous system, and bone metastasis). EGFR was evaluated in 99 patients and considered positive in 53% primary tumors. In 36% of primary tumors expressing EGFR, the corresponding metastatic site was found negative, whereas it was found positive in 15% of patients from EGFR negative primary cancers. Overall survival (%) Positive expression Negative expression p=0.004 months 1066 Int J Colorectal Dis (2009) 24:1059–1068 Table 7 Univariate analysis related to EGFR, ErbB2, ErbB3, and ErbB4 expression and overall survival Variable Expression EGFR Negative Positive Negative Positive Negative Positive Negative Positive Negative Positive ErbB2 ErbB3 ErbB4 (membrane) ErbB4 (cytoplasmic) 5-year survival (%) p value 76.1 65.1 71.0 55.6 51.5 77.6 70.1 66.7 68.2 76.2 0.24 0.36 0.004 0.31 0.55 Goldstein and Armin [29] reviewed 102 patients with colorectal cancer and observed EGFR expression in 75.5% of cases. They analyzed the hypothesis that EGFR expression may change during the process of cancer invasion and found correlation with the risk of recurrence and death when EGFR was expressed in the deep regions of colorectal cancers. Other authors investigated the prognostic significance of EGFR expression in colorectal cancers. Spano et al. [26] examined tumor samples from 150 patients and found EGFR overexpression in 80% of cases and it was associated with TNM stage T3, but with no survival correlation. In the other hand, Steele et al. [30] observed in 50 patients significant EGFR expression in stage III and poor differentiated tumors, with poor correlation to survival. This fact was also seen in Galizia et al. [31] study of 126 colon cancer patients, submitted to curative surgery. EGFR expression were detected in 35.6% of patients and was correlated with disease recurrence and worse survival in both univariate and multivariate analysis. In the current study, positive expression of EGFR was found in 57.8% of patients and, as in most of the series, there was no correlation with recurrence and overall survival. Table 8 Association between ErbB3 expression and the risk of death (multivariate analysis) Variable HR 95% CI p value Presence of lymphatic invasion ErbB3 negative expression ErbB4 positive membranous expression Presence of perineural invasion 1.81 2.46 1.79 0.89–3.68 1.19–5.08 0.60–5.33 0.10 0.014 0.29 1.69 0.77–3.70 0.18 Estimated risk from Cox regression model with matching variables lymphovascular invasion, ErbB3 expression, ErbB4 expression, and perineural invasion HR hazard ratio, CI confidence interval The ErbB2 expression was evaluated by some series and the majority could not find any correlation with its expression and prognosis. Nathanson et al. [32] showed correlation between gene amplification and immunohistochemical expression of ErbB2 in 139 patients with colon cancer. Only 3.6% of tumors expressed ErbB2 and there was no association either with stage or with survival. Schuell et al. [33] and Ochs et al. [34] also found membranous expression in a few primary colorectal tumors, respectively 30% and 11% of 77 and 109 patients. McKay et al. [35] evaluated ErbB2 expression in 249 primary colorectal tumors and 42 lymph node metastasis. They found positive expression in 81.8% of patients with concordant staining with lymph node metastasis in only 52.4% of cases. Colon cancers were more likely to express ErbB2 than rectal cancers. They also showed no association with gene polymorphism and no correlation with survival. The cytoplasmic expression of ErbB2 was evaluated by two series, with conflicting results. Essapen et al. [36] noted in 170 colorectal tumors membranous expression in 41% and cytoplasmic expression in 87% of the patients. The cytoplasmic expression was related to better prognosis in stage III patients. However, Osako et al. [37] reported in 146 colorectal tumors membranous expression in 3% and cytoplasmic expression in 68.5% of the cases. The cytoplasmic expression had correlation with worse overall survival in stage II patients. In the current study, we found ErbB2 membranous positive expression in only 8.3% of cases. As in EGFR expression, our series found no association between the positive expression of ErbB2 and the increase risk of recurrence and death. Other studies evaluated the coexpression of EGFR and ErbB2. Kluftinger et al. [38] showed co-expression in 17% of 35 cases and Cunningham et al. [39] in 75% of 87 patients. The latter found EGFR and ErbB2 cytoplasmic expression in 63% and 75% of cases. In our study, we found co-expression of EGFR and ErbB2 in only 7.9% of the patients. In CRC, only a few studies investigated the protein expression of ErbB3 and ErbB4. Maurer et al. [40] revealed an ErbB3 expression rate of 89% in colorectal tumors both membranous and cytoplasmic, with co-expression with ErbB2 in 77% of the cases. Kapitanovic et al. [41] demonstrated that ErbB3 is expressed in 78% of cases, and was exclusively cytoplasmic. Until now, Lee at al. [42] published the only article that had examined the expression of all ErbB family proteins in patients with colorectal cancer. Only membranous staining was considered positive. They evaluated 125 patients submitted to curative surgery and considered EGFR, ErbB2, ErbB3, and ErbB4 positive expression respectively in 52%, 35%, 36%, and 22% of cases. A significantly higher percentage of overexpressed ErbB3 was observed in early stage carcinomas (stages I and II). Int J Colorectal Dis (2009) 24:1059–1068 However, ErbB4 was more overexpressed in advanced stage cancers (stages III and IV). The expression of EGFR, ErbB2, ErbB3, and ErbB4 alone denoted no association with a shortened survival, but patients with simultaneous overexpression of ErbB2 and ErbB4 had a shorter overall survival time in only the univariate analysis, probably because of correlation with more advanced stages. Kountourakis et al. [43] evaluated the ErbB3 and ErbB4 expression in 106 colorectal tumors. Both membranous and cytoplasmic expression was identified. The rates of expression for ErbB3 and ErbB4 respectively were 17% and 18.9% membranous; 28.3% and 30.2% cytoplasmic. ErbB3 positive cytoplasmic expression was associated with moderate tumor grade and ErbB4 membranous expression was associated with lymph node involvement. There was no correlation between the ErbB3 and ErbB4 expression and patient prognosis. In our series, negative expression of ErbB3 was significantly associated with the presence of lymphovascular invasion. However, ErbB3 negative expression maintained in multivariate analysis, the negative impact both in risk of recurrence and death. ErbB3 protein has low affinity to ligands when compared to other receptors of ErbB family. The low expression may indirectly increase or perpetuate other signaling pathways. Another hypothesis is the possibility that the cytoplasmic sequestering of ErbB3 receptors may alter the propensity to form ErbB3-containing heterodimers at the level of plasma membrane [44]. There is large evidence that ligand-induced heterodimerization of ErbB family receptors is necessary to diversify and specify biological responses. The role of ErbB3 protein to act as an efficient suppressor of ligand-induced ErbB4 signaling was also described [45] and the overexpression of ErbB3 somehow may inhibit signaling transcription induced by ErbB family receptors ligands. As previously mentioned, positive membranous expression of ErbB4 influenced the risk of recurrence both in univariate and multivariate analysis. There was no correlation to overall survival. The cytoplasmic expression of ErbB4 had no prognostic correlation to relapse and death. There are several possible reasons for discrepancies between studies. The small sample size, the disparate scoring systems used to classify family ErbB overexpression, the source of antibody used, and the immunohistochemical protocol make comparison between the studies very challenging. Scoring systems for tumor markers in CRC are usually based on a measure of proportion of positive tumor cells and combined with a degree of staining intensity [46]. It is recognized that the interpretation of staining intensity is not only highly subjective but may be affected by storage time, variation in protocols, and fixation procedures. Despite these concerns, staining intensity has become an integral 1067 component of many immunohistochemical scoring methods for tumor markers in colorectal cancer. Goethals et al. [47] has reported that four cores biopsies are sufficient to account for tumor heterogeneity. Although it is argued that a single core sample (0.6 mm) per tumor may not be representative of the whole tumor, results even using one sample tumor have shown well-established associations between molecular features and clinico-pathological endpoints [48, 49]. TMA technology allowed us to analyze 415 tumor samples. Ninety three patients had four tumor samples, 11 patients had three tumor samples, and five patients had two tumor samples. We had no primary tumor sampled with one core. 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