Expression And Prognostic Role Of ABCG2 In Colorectal Cancer

Background and aimsColorectal cancer is the third most common cause of death from cancer in both sexes in the western world and the high incidence is arising in China. Although this disease is surgically curable in early stages, the tumor frequently does not become symptomatic before the metastatic stage, which is associated with high mortality. Therefore, increasing efforts are being made to develop more effective screening and prevention strategies for colorectal cancer and to enhance our capabilities to predict the clinical. Conventional prognostic and predictive markers for patient survival are histologic tumor grade and tumor stage at the time of diagnosis (TNM or Dukes), including depth of tumor invasive, involvement of regional lymph nodes, and metastatic spread to distant organs. In addition to these clinicopathologic variables, molecular markers are being evaluated and established for a wide variety of tumors including colon cancer. These biomarkers can identify patients with a high risk of recurrence or metastases who may be difficult to identify using traditional clinicopathologic indexes.ABCG2, also known as breast cancer resistance protein (BCRP)/placental ABC protein (ABCP)/mitoxantrone resistance protein (MXR), is a member of the superfamily of ATP-binding cassette (ABC) transporter proteins, which use the energy of ATP hydrolysis to transport a wide variety of substrates across the cell membrane. ABCG2 is well known for its capacity to efflux an array of therapeutic compounds, such as mitoxantrone, toptecan, irinotecan, flavopiridol, methotrexate, which leads to drug resistance and treatment failure. ABCG2 also efflux Hoechst 33342, and is known to be a molecular determinant of the side population phenotype, characteristics of which is reminiscent of stem cells. Moreover, previous studies suggest that ABCG2 protects cells from hypoxia-related cell damage by reducing the accumulation of toxic heme metabolites, indicating a protective function of ABCG2 in stem cells.As a biomarker, the expression of ABCG2 has been reported to be an unfavorable prognostic factor in acute myeloid leukemia, adult acute lymphoblastic leukemia, non-small cell lung cancer, small-cell lung cancer, and esophageal squamous cell carcinoma. A study of 13 patients by Gupta et al found high expression of BCRP in the apical surface of normal small bowel and colon epithelial cells., however pretreatment specimens of colon cancer and a hepatic metastasis had lower BCRP mRNA and protein expression compared with normal colon. Glasgow et al. studied BCRP mRNA expression in 21 mucinous compared with 30 nonmucinous Dukes C colon cancer specimens. Although mucinous tumors have a worse prognosis than nonmucinous ones, there was no difference in BCRP mRNA expression between mucinous and nonmucinous tumors. However, no data on the relevance of ABCG2 expression and its clinical significance including patient survival time in colorectal carcinoma.We aimed to investigate the expression of ABCG2 in larger tumor samples of colorectal carcinomas and to evaluate its prognostic significance.Materials and Methods1. Patients Two hundred twenty-five consecutive cases (age,19 to 83 years; median,57) who were diagnosed for colorectal cancer at the Institute of Pathology, Huizhou Municipal Central Hospital, between 2003 and 2005 were enclosed in this study. Only patients with primary colorectal adenocarcinomas without neoadjuvant radiochemotherapy and no other known malignancies at the time of diagnosis were included. Cancer tissue specimens were collected from the patients after informed consent had been obtained. Histologic diagnosis was established on standard H&E-stained sections according to the guidelines of the WHO. Clinical follow-up data were available for 69 of the 225 patients (30.7%); those without complete data were excluded from further analyses. Twenty-seven (39.1%) patients died during follow-up with a median survival of 12.1 months.2. ImmunohistochemistryFormalin-fixed paraffin-embedded tissue was freshly cut (4μm).The sections were mounted on superfrost slides, deparaffinized in xylene and grehydrated through graded ethanols. Before staining, antigen retrieval was achieved by microwave oven in 0.01 mol/L citrate buffer (PH=6.0) for 10 min and slides were cooled to room temperature. Endogenous peroxidase activity was blocked using 0.3% H2O2 in methanol for 20 min. Nonspecific binding was blocked by incubation with 5% normal goat serum/PBS for 20 min. The primary ABCG2 antibody (BXP-21, Abcam, England) was diluted 1:40 using a background reducing dilution buffer(maixin,China) and was incubated at 4℃overnight. Detection took place by the conventional labeled streptavidin biotin method (LSAB kit,).3,3-Diaminobenzidine tetrahydrochloride was used as the chromogen. The secondary antibody was goat anti-mouse IgG. Sections were then briefly counterstained with haematoxylin and mounted.All immunostained slides were inspected by two independent clinical pathologists who were unaware of patient outcome. As ABCG2 showed a cytoplasmic and membranous staining, we scored both staining qualities as either negative or weakly, moderately, or strongly positive. Firstly, the intensity of the ABCG2 immunostaining was scored as follows:0, no color; 1,yellow; 2, brown; 3, dark brown. Then the number of ABCG2 positive cells was scored as follows: 1,<=10% positive cells; 2,11-30% positive cells; 3,31-50% positive cells; 4, >50%. These two scores were multiplied together. Those 4 scores were defined 0 as-;1～3 as+; 4 as++;>=5 as+++, here-was defined as negative,+was; defined as weakly positive,++was defined as moderately positive,and+++ were defined was defined as strongly positive.3. Statistical analysisThe data were compiled with the software package SPSS, version 13.0. The Spearman rank-order correlations coefficient and non-parametric Kruskal-Wallis or Mann-Whitney tests were employed to test the significance of associations between expression of ABCG2 and clinicopathologic variables. Univariate survival analysis was done according to Kaplan-Meier, and differences in overall survival among subgroups were compared by using the log-rank test. Multivariate survival analysis was done on all variables that were found to be significant on univariate analysis using Cox's regression model. P< 0.05 was considered significant.Results1. ABCG2 immunohistochemistry and statistical analysisAbout 50% normal colon mucosa exhibited a strong membranous staining on the apical membrane. And some cases showed that small blood vessels, within connective tissue and intratumoral areas, exhibited prominent ABCG2 expression. In general, colorectal carcinomas showed high levels of ABCG2 expression for cytoplasmic ABCG2 staining,83.1% of tumor were positive and 13.3% were strongly positive. Higher rates of cytoplasmic ABCG2 immunoreactivity were associated with higher Dukes stage, but showed no significant correlation to other clinicopathologic variables. For membranous ABCG2 staining,66.2% of tumor were positive and 15.6% were strongly positive. Membranous ABCG2 immunoreactivity was significantly associated with higher tumor stage Dukes, node and systemic metastasis.2. Survival analysisClinical follow-up data were available for 69 of the 225 patients (30.7%). Cases with a strong cytoplasmic and membranous ABCG2 positivity showed a significantly shortened mean survival time in comparison with the other patients (P<0.0001,P=0.001).Other prognostic factors in univariate survival analysis were Dukes stage, nodal status and state of distant metastasis.In the multivariate survival analysis, membranous ABCG2 expression remained a significant independent prognostic factor (P= 0.038) with a relative risk of 2.439 (confidence interval,1.053-5.650). Other independent prognostic factor in multivariate survival analysis was the extent of tumor systemic metastasis. The prognostic value of strong cytoplasmic ABCG2 expression failed significance in the multivariate survival analysis.ConclusionsABCG2 is commonly up-regulated in colorectal cancer. Strong membranous ABCG2 expression was significantly associated with higher tumor stages (Dukes), lymph node metastasis, systemic metastasis and prognosis. Univariate and multivariate analyses revealed that membranous ABCG2 expression is an independent prognosis factor of overall survival which corroborates the importance of ABCG2 in tumor progression of this disease.