Colorectal cancer is considered to be one of the most common human malignancies. It affects about one million individuals worldwide every year (El-Tawil, 2010). In Egypt, it is the sixth most common cancer. It represents 4% of total cancers in both sexes (Zeeneldin et al., 2012).
Both genetic susceptibility and environmental influences contribute toward the development of colorectal cancer (Lyall et al., 2006). About 5% of adenomatous polyps in the large bowel progress to malignant tumors within 5–10 years (Weitz et al., 2005).
Earlier diagnosis of colorectal cancer, determination of its clinicopathological prognostic factors, and surgical treatments associated or not with chemotherapy have improved the outcome of affected patients (Spano et al., 2005).
Researchers have attempted to identify biological markers in order to individualize chemotherapy and to predict clinical outcome and tumor sensitivity (Nanni et al., 2002).
Galectin-3 (Gal-3) is B-galactoside-binding animal lectin that contains carbohydrate-recognition domains (Lee et al., 2009). It is a 31 kDa gene product that acts as intracellular and extracellular lectin and can interact with glycoproteins located on the cell surface matrix (Sakaki et al., 2010). It plays important roles in cell adhesion, proliferation, differentiation, cell cycle progression apoptosis, and immune system regulation in addition to its involvement in tumor genesis, angiogenesis, and tumor metastasis (Lee et al., 2009; Zhang et al., 2009; Sakaki et al., 2010).
The importance of Gal-3 expression was evaluated in many types of neoplasm. It could be a reliable diagnostic marker and one of the target proteins in cancer treatment. It was upregulated in cancers of the thyroid, liver, stomach, and tongue and was downregulated in ovarian, uterine, and breast cancers. However, in colon cancer, conflicting results have been obtained (Legendre et al., 2003; Arfaoui-Toumi et al., 2010).
Gal-3 can be used as a prognostic factor to predict poor outcome of patients with colorectal carcinoma as it plays an important role in its progression (Arfaoui-Toumi et al., 2010).
c-erbB-2 is a proto-oncogene that encodes a transmembrane protein with tyrosine kinase activity. It is closely related to EGFR but distinct from it biologically. Amplification of the c-erbB-2 gene or overexpression of its protein has been found in several tumors such as breast, stomach, lung, and urinary bladder tumors. Several studies have reported the value of c-erbB-2 in predicting the biological behavior of tumors (Ghaffarzadegan et al., 2006).
The aim of this study was to evaluate the role of both Gal-3 and c-erbB-2 in colorectal tumors in relation to early detection and prognosis and also to study their relationship with the main clinicopathological factors.
Materials and methods
Fifty paraffin wax-embedded specimens of colorectal tumors were available in our study, 35 with colorectal carcinoma and 15 with colorectal adenoma, of which nine cases were tubular adenoma and six were tubulovillous adenoma. All cases were retrieved from the files of the Histopathology Department, Faculty of Medicine, Zagazig University Hospital and private clinics in the period between 2000 and 2009. Among the patients with colorectal cancer, there were 22 men and 13 women and among those with adenoma, there were 11 men and four women. The age range was 28–76 years, with a median age of 65 years.
A section from specimen blocks was stained with hematoxylin and eosin for histological evaluation. The carcinoma cases were graded and staged according to the WHO grading system and Duke’s classification, respectively. Representative blocks were chosen for immunostaining.
Two consecutive 4-μm-thick sections were cut, mounted on positively charged slides, and immunostained with monoclonal antibody to Gal-3 (diluted 1 : 50) and for c-erbB-2 immunostaining diluted at 1 : 50. Both antibodies were obtained from Dakopaths (Gloftrup, Denmark).
The avidin–biotin complex method was used for immunostaining. The sections were dewaxed and treated with 0.3% hydrogen peroxide (H2O2) in methanol for 30 min to block the endogenous peroxidase activity. The slides were rinsed in distilled water and incubated for 40 min at 99°C in a water bath with 10 ml of citrate buffer.
The primary antibodies were applied to the sections and incubated at 4°C. This was followed by incubation with biotin-labeled anti-mouse secondary antibodies for 30 min and ABC for 60 min. Careful rinses were performed with PBS between each step of the procedure. The color was developed with diaminobenzidine and the sections were counterstained with hematoxylin, dehydrated, and mounted. Sections from adjacent healthy mucosa were stained immunohistochemically for both markers and used as negative control slides.
Evaluation of Galectin-3 immunostaining
The staining patterns were graded as membranous and/or cytoplasmic.
The staining intensity was scored as follows: negative (0), weak (1+), moderate (2+), and strong (3+).
Positive staining was defined as 2+ to 3+ staining intensity in more than 10% of the cells (Scognamiglio et al., 2006).
Evaluation of c-erbB-2 immunostaining
c-erbB-2 staining was noted as membranous staining. Cases were considered positive when at least 10% of cells showed positive staining with the relevant antibody.
The expression was graded as follows: <10% (negative), 10–40% (+), 41–70% (++), >70% (+++) (Ghaffarzadegan et al., 2006).
The relationship between Gal-3 and c-erbB-2 expression and the clinicopathological variables were analyzed with χ 2-test, and a P value less than 0.05 was considered significant.
Our analysis included 35 specimens of colorectal carcinoma and 15 specimens of colorectal adenoma. The adenomas were tubular in nine cases (60%) and tubulovillous in six (40%). Among patients with colorectal adenocarcinoma, there were 22 men (62.9%) and 13 women (37.1%), with 16 (45.7%) patients younger than 50 years of age and 19 (54.3) patients older than 50 years of age . Most of the adenocarcinoma (60%) was located in the colon and 40% in the rectum. The tumor size was less than 5 cm in 11(31.4%) cases and at least 5 cm in 24 (68.6%) cases. GI tumor differentiation was found in 16 (45.7%) cases, whereas GIII was found only in seven (20%). Duke’s classification class A was present in 13 (37.1%) cases, whereas class D was present only in five (14.3%) (Table 1).
Galectin-3 staining results
The current study showed membranous and/or cytoplasmic brown staining of neoplastic cells with complete negativity in the adjacent normal mucosa. Gal-3 immunostaining was predominant in colorectal cancer cases, 91.4% (32/35), than in adenoma cases, 33.3% (5/15) (P<0.001) (Table 2). In terms of the grade of differentiation and Duke’s stage, Gal-3 expression was greater in cases of high histological grade GIII, 100% (7/7) (P=0.009), and those with advanced Duke’s stage (100%, P=0.014). No significant association was found between Gal-3 expression and patients’ age, tumor size, or tumor location (P=0.76, 0.57, and 0.47, respectively) (Table 3).
Gal-3 staining intensity
In adenoma cases, only one case of tubulovillous adenoma (16.7%) showed a strong (3+) staining pattern (Fig. 2), and moderate (2+) staining was noted in two other cases of tubulovillous adenoma (13.3%). However, tubular adenoma showed a weak (1+) staining pattern in two cases (22.2%) (Fig. 1). A negative reaction was noted in the remaining cases, 66.7% (10/15).
In carcinoma cases, 8.6% (3/35) of cases had no reactivity, 25.7% (9/35) had weak (1+) reactivity, 28.6% (10/35) had moderate (2+) reactivity, and 37.1% (13/35) showed strong (3+) reactivity (Figs 3 and 4).
Thus, there was a highly significant association between high grade of Gal-3 positivity (2+, 3+) and carcinoma cases (P<0.001; Table 4).
c-erbB-2 immunostaining pattern
The staining pattern of c-erbB-2 was predominantly membranous. The adjacent normal mucosa showed a low c-erbB-2 expression (in <10% of cells).
In adenoma, most cases (93.3%, 14/15) were positive and only one case (6.7%, 1/15) showed negative c-erbB-2 expression.
In carcinoma cases, the overall incidence of c-erbB-2 expression was observed in 65.7% (23/35) (P=0.04) (Table 2). In our study, c-erbB-2 immunoreactivity was inversely associated with tumor grade and stage as it was highly expressed in 87.5% (14/16) of low-grade tumor GI (P=0.019) and in 92.3% (12/13) of tumors with early stage A (P=0.0016) (Table 3).
Also, we found an association between c-erbB-2 expression and the primary site of the tumor as it was expressed in rectal cases (85.7%, 12/14) more than that in colon cancer cases (52.4%, 11/21) (P=0.04; Table 3).
However, no significant association was found between its expression and patients’ age or tumor size (P=0.14 and 0.45), respectively.
In terms of the staining intensity of c-erbB-2, there was a significant association between c-erbB-2 strong reactivity (>70%) and adenoma cases (46.7%, 7/15), rather than carcinoma cases (17.1%, 6/35) (P=0.02) (Table 5 and Figs 5–7).
Colorectal cancer is considered one of the leading causes of cancer deaths worldwide. The discovery of new prognostic markers in patients with colorectal cancer can improve the outcome of disease and define new therapeutic approaches for these patients (Spano et al., 2005).
Gal-3 is a 30 kDa protein. It is widely distributed in normal adult tissues, especially the epithelial cells and cells involved in immune response. It was also found to be expressed in a variety of tumors (Eude-le Parco et al., 2009).
Most of the studies carried out on tumor cell lines have reported that Gal-3 may be involved at various levels of the tumorigenic cascade (Nakahara and Raz, 2007).
It was upregulated in cancers of the thyroid, liver, stomach, and tongue and was downregulated in ovarian, uterine, and breast cancers. However, in colon cancer, the results were conflicting (Legendre et al., 2003; Arfaoui-Toumi et al., 2010).
In our study, Gal-3 showed high significant overexpression in colorectal cancer cases (91.4%) more than in adenomatous cases (33.3%) (P<0.001). It was strongly expressed in only one case of tubulovillous adenoma (3+) and moderately (2+) expressed in other two cases. This was similar to the result reported by Greco et al. (2004), who found that Gal-3 overexpression was associated with tubulovillous lesions.
However, we noted that Gal-3 staining intensity was significantly associated with a high grade of differentiation (GIII) of adenocarcinoma cases. This was similar to the study by Endo et al. (2005); however, Arfaoui-Toumi et al. (2010) noted a progressive decrease in Gal-3 staining intensity with decreasing degree of tumor differentiation (GIII).
In terms of staging, some studies showed that overexpression of Gal-3 is associated with advanced tumor stage and shortened survival (Guévremont et al., 2004; Goh et al., 2005). In contrast, other studies have reported decreased levels of Gal-3 expression in cancer colon progression (Lotz et al., 1993; Sablin et al., 2009).
The current study showed a significant association between high grade of Gal-3 staining intensity and the advanced stage of Duke’s classification. Endo et al. (2005) also found that Duke’s stage C and D showed higher expression of Gal-3 more than stages A and B. This was in agreement with Wu and Gan (2007), who concluded that Gal-3 expression was higher in tumors with lymph node metastasis (higher stages) than tumors without metastasis.
Findings similar to those of our study were obtained by Zhang et al. (2006), who reported that Gal-3 expression was correlated positively with poor differentiation, invasion, and metastasis of colorectal carcinoma.
In this work, no significant association was found between Gal-3 expression and patients’ age, tumor size, or tumor location (P=0.76, 0.57, and 0.47), respectively. In contrast, Endo et al. (2005) found a correlation between Gal-3 expression and larger size tumors.
Many recent studies have reported that Gal-3 immunoexpression plays an important role in colorectal cancer progression and can be used as a prognostic factor to predict poor outcome of patients in addition to identifying new targeted therapy to improve the prognosis of patients with colorectal cancer (Arfaoui-Toumi et al., 2010; Zaia Povegliano et al., 2011).
The human epidermal growth factor receptor-2 (HER-2 or c-erbB-2) is a member of the tyrosine kinase receptor super family (Kavanagh et al., 2009). Its proto-oncogene is frequently expressed at low levels in many human adult epithelial cells and amplified or overexpressed in carcinoma of glandular epithelial origin. Thus, its expression along with other growth factor receptors is considered to play a role in the pathogenesis of colorectal carcinoma (Porebska et al., 2000).
In our study, c-erbB-2 was expressed in adenomas and carcinomas with a weak expression in the adjacent normal mucosa. It was significantly highly expressed in adenoma than carcinoma as the percentage of stained cells was 93.3% in adenoma and 65.7% in carcinoma (P=0.04). Also, the degree of staining intensity of c-erbB-2 showed +3 intensity in adenomatous cases more than in carcinomatous ones (P=0.02).
Similar results were obtained by Uner et al. (2005), who found that c-erbB-2 expression was detectable more frequently in adenoma than in carcinoma. Also, Porcelli et al. (2001) reported that its expression was significantly higher (P<0.01) in adenomatous lesions than in colorectal cancer.
In contrast to our results, Kapitanovic et al. (1997) found that c-erbB-2 was significantly overexpressed in colorectal carcinoma more than in benign and premalignant lesions with intense mixed membrane and cytoplasmic staining of tumor cells and negative surrounding normal tissue.
In our study, we found an inverse association between c-erb-2 overexpression and tumor grade and stage, as it was associated with low-grade tumors (P=0.019) and early-stage tumors (P=0.0016). This was in agreement with the results of other studies (Porcelli et al., 2001; Jin et al., 2004). Also, the study by McKay et al. (2002) found no relation between patient survival and c-erbB-2 and concluded that c-erbB-2 does not play a prognostic role in colorectal cancer.
In relation to primary tumor location, the present study showed a significant association between c-erbB-2 expression and rectal location of tumors. This result was similar to that obtained by Demirbaş et al. (2006), and was in contrast to that found by McKay et al. (2002).
No significant association was found between c-erbB-2 expression and patient age or tumor size (P=0.14 and 0.45, respectively) in this study. These results was similar to those of many other studies (Nathanson et al., 2003; Schuell et al., 2006).
- c-erbB-2 protein overexpression occurs in adenomas more than carcinomas, and it is inversely associated with the grade and the stage of colorectal cancer. Thus, it can be considered as a predictor of the occurrences of colorectal carcinoma and not as a prognostic marker.
- Gal-3 can predict cancer behavior and clinical outcome in patients with colorectal cancer; thus, it can be considered as a prognostic marker.
- Selection of colorectal adenocarcinoma patients with Gal-3 overexpression for treatment with anti-Gal-3 target therapy is considered a new era in the treatment of colorectal cancer.
Conflicts of interest
There are no conflicts of interest.
Arfaoui-Toumi A, Mahmoud LK-B, Hmida MB, Khalfallah M-T, Regaya-Mzabi S, Bouraoui S.Implication of the galectin-3 in colorectal cancer development (about 325 Tunisian patients).Bull Cancer2010;97:E1–E8.
Demirbaş S, Sücüllü I, Yildirim S, Çelenk T.Influence of the c-erb B-2, nm23, bcl-2 and p53 protein markers on colorectal cancer.Turk J Gastroenterol2006;17:13–19.
El-Tawil AM.Colorectal cancer and pollution.World JGastroenterol2010;16:3475–3477.
Endo K, Kohnoe S, Tsujita E, Watanabe A, Nakashima H, Baba H, Maehara Y.Galectin-3 expression is a potent prognostic marker in colorectal cancer.Anticancer Res2005;25:3117–3122.
Eude-le Parco I, Gendronneau G, Dang T, Delacour D, Thijssen VI, Edelmann W, et al..Genetic assessment of the importance of galectin-3 in cancer initiation, progression, and dissemination in mice.Glycobiology2009;19:68–75.
Ghaffarzadegan K, Sharifi N, Vosooghynia H, Shakeri T, Ghiasi Moghadam T, Ghanad Kafi Sh, et al..HER2/neu expression in colon adenocarcinoma and its correlation with clinicopathological variables.Spring, IJBMS2006;9:64–69.
Goh K-L, Quek K-F, Yeo GTS, Hilmi IN, Lee C-K, Hasnida N, et al..Colorectal cancer in Asians: a demographic and anatomic survey in Malaysian patients undergoing colonoscopy.Aliment Pharmacol Ther2005;22:859–864.
Greco C, Vona R, Cosimelli M, Matarrese P, Straface E, Scordati P, et al..Cell surface overexpression of galectin-3 and the presence of its ligand 90k in the blood plasma as determinants in colon neoplastic lesions.Glycobiology2004;14:783–792.
Guévremont M, Martel-Pelletier J, Boileau C, Liu F-T, Richard M, Fernandes J-C, et al..Galectin-3 surface expression on human adult chondrocytes: a potential substrate for collagenase-3.Ann Rheum Dis2004;63:636–643.
Jin W, Gao M-Q, Lin Z-W, Yang D-X.Multiple biomarkers of colorectal tumor in a differential diagnosis model: a quantitative study.World J Gastroenterol2004;10:439–442.
Kapitanovic S, Radosevic S, Kapitanovic M, Andelinovic S, Ferencic Z, Tavassoli M, et al..The expression of p185(HER-2/neu) correlates with the stage of disease and survival in colorectal cancer.Gastroenterology1997;112:1103–1113.
Kavanagh DO, Chambers G, O’Grady L, Barry KM, Waldron RP, Bennani F, et al..Is overexpression of HER-2 a predictor of prognosis in colorectal cancer?BMC Cancer2009;9:1.
Lee J, Moon C, Kim J, Jung C, Lee KH, Joo HG, et al..Immunohistochemical localization of galectin-3 in the granulomatous lesions of paratuberculosis-infected bovine intestine.J Vet Sci2009;10:177–180.
Legendre H, Decaestecker C, Nagy N, Hendlisz A, Schüring M-P, Salmon I, et al..Prognostic values of galectin-3 and the macrophage migration inhibitory factor (MIF) in human colorectal cancers.Mod Pathol2003;16:491–504.
Lotz MM, Andrews CW Jr, Korzelius CA, Lee EC, Steele GD Jr, Clarke A, Mercurio AM.Decreased expression of Mac-2 (carbohydrate binding protein 35) and loss of its nuclear localization are associated with the neoplastic progression of colon carcinoma.Proc Natl Acad Sci USA1993;90:3466–3470.
Lyall MS, Dundas SR, Curran S, Murray GI.Profiling markers of prognosis in colorectal cancer.Clin Cancer Res2006;12:1184–1191.
McKay JA, Murray LJ, Curran S, Ross VG, Clark C, Murray GI, et al..Evaluation of the epidermal growth factor receptor (EGFR) in colorectal tumours and lymph node metastases.Eur J Cancer2002;38:2258–2264.
Nakahara S, Raz A.Regulation of cancer-related gene expression by galectin-3 and the molecular mechanism of its nuclear import pathway.Cancer Metastasis Rev2007;263–4605–610.
Nanni O, Volpi A, Frassineti GL, De Paola F, Granato AM, Dubini A, et al..Role of biological markers in the clinical outcome of colon cancer.Br J Cancer2002;87:868–875.
Nathanson DR, Culliford AT IV, Shia J, Chen B, D’Alessio M, Zeng Z-S, et al..HER 2/neu expression and gene amplification in colon cancer.Int J Cancer2003;105:796–802.
Porcelli B, Frosi B, Terzuoli L, Arezzini L, Marinello E, Vernillo R, et al..Expression of p185 and p53 in benign and malignant colorectal lesions.Histochem J2001;33:51–57.
Porebska I, Harłozińska A, Bojarowski T.Expression of the tyrosine kinase activity growth factor receptors (EGFR, ERB B2, ERB B3) in colorectal adenocarcinomas and adenomas.Tumor Biol2000;21:105–115.
Sablin M-P, Italiano A, Spano J-P.Colorectal cancers: prognostic and predictive factors of response to treatment.Bull Cancer2009;96:417–423.
Sakaki M, Fukumori T, Fukawa T, Elsamman E, Shiirevnyamba A, Nakatsuji H, Kanayama H-O.Clinical significance of Galectin-3 in clear cell renal cell carcinoma.J Med Invest2010;571–2152–157.
Schuell B, Gruenberger T, Scheithauer W, Zielinski Ch, Wrba F.HER 2/neu protein expression in colorectal cancer.BMC Cancer2006;6:123.
Scognamiglio T, Hyjek E, Kao J, Chen Y-T.Diagnostic usefulness of HBME1, galectin-3, CK19, and CITED1 and evaluation of their expression in encapsulated lesions with questionable features of papillary thyroid carcinoma.Am J Clin Pathol2006;126:700–708.
Spano J-P, Lagorce C, Atlan D, Milano G, Domont J, Benamouzig R, et al..Impact of EGFR expression on colorectal cancer patient prognosis and survival.Ann Oncol2005;16:102–108.
Uner A, Ebinc FA, Akyurek N, Unsal D, Mentes BB, Dursun A.Vascular endothelial growth factor, C-ERBB-2 and C-ERBB-3 expression in colorectal adenoma and adenocarcinoma.Exp Oncol2005;27:225–228.
Weitz J, Koch M, Debus J, Höhler T, Galle PR, Büchler MW.Colorectal cancer.Lancet2005;365:153–165.
Wu ZH, Gan L.Association of galectin-3 and E-cadherin expressions with lymph node metastasis of colon cancer.Nan Fang Yi Ke Da Xue Xue Bao2007;27:1731–1733.
Zaia Povegliano L, Oshima CT, de Oliveira Lima F, Andrade Scherholz PL, Manoukian Forones N.Immunoexpression of galectin-3 in colorectal cancer and its relationship with survival.J Gastrointest Cancer2011;42:217–221.
Zeeneldin AA, Saber MM, Seif El-din I, Farag SA.Colorectal carcinoma in Gharbiah District, Egypt: comparison between the elderly and non-elderly.J Solid Tumors2012;2:13–23.
Zhang H-Y, Jin L, Stilling GA, Ruebel KH, Coonse K, Tanizaki Y, et al..RUNX1 and RUNX2 upregulate Galectin-3 expression in human pituitary tumors.Endocrine2009;35:101–111.
©2013Egyptian Journal of Pathology
Zhang N, Ding YQ, Liang L.Association of galectin-3 expression with biological behaviors of human colorectal carcinoma.Nan Fang Yi Ke Da Xue Xue Bao2006;26:1685–1689.