Impact of ABCB1 and ABCG2 Transporter in Outcome of Gallbladder Cancer

Abstract

Background: Gallbladder cancer (GBC) is a biologically aggressive malignancy requiring appropriate biomarkers to improve its outcome. Role of ABC transporters (ABCB1 and ABCG2) has been linked to cancer aggressiveness, tumorigenesis and multidrug resistance. Herein, we studied the prognostic implication of ABCB1 and ABCG2 in GBC.

Methods: Fresh tissue (tumour & normal) samples collected from 54 patients who underwent R0 resection, were analysed for mRNA and protein expression of ABCB1 and ABCG2 by quantitative Real-Time PCR and western blotting, respectively, in this prospective study. The molecular findings were correlated with clinical-pathological parameters using χ2 and fisher exact test. The molecular changes in ABCB1 and ABCG2 were analysed for predicting overall survival (OS), disease-free survival (DFS) and response to chemotherapy using Kaplan-Meier log-rank test and Cox regression multivariate analysis.

Results: The mean age of the cohort was 50 ± 13.2 with 26 (48.1%) in patients having early stage gallbladder cancer (GBC). Over-expression of ABCB1 and ABCG2 was noted in 32/54 (59%) and 40/54 (74%) cases, respectively. The protein expression of ABCB1(P-glycoprotein) and ABCG2 (BCRP) was higher in 27/54 (50%) and 37/54 (59%) cases, respectively. The mean OS and DFS was 20.7 ± 11.5 and 19.3 ± 12.2 months at median follow-up of 24 months. The TNM stage, lymph node metastasis, and presence of gallstone were significant factors for predicting OS and DFS on multivariate analysis. Both ABCB1 and ABCG2 did not show any significant correlation with OS and DFS with similar incidences of late death and recurrence among over-expression and down-expression. Sub-group comparison suggests that change in expression pattern of ABCB1 and ABCG2 may not affect response to chemotherapy in GBC.

Conclusion: Altered expression of ABCB1 and ABCG2 may not be a useful prognostic marker for survival or response to chemotherapy in GBC. Presently, histo-pathological characteristics and associated gallstones are the important predictors for survival and recurrence in GBC.

Keywords: ABC transporters; ABCB1; ABCG2; chemotherapy; gallbladder cancer.

Figure 1

Box-plot representation of mRNA expression; a) Normalised expression (2 ˆ ^ΔCt) of ABCB1 in tumour and normal tissue; b) Normalised expression (2 ˆ ^ΔCt) of ABCG2 in tumour and normal tissue c) Relative fold increase (2 ˆ ^-ΔΔCt) of ABCB1 and ABCG2 in tumour with respect to normal tissue in GBC patients. {The thick line consists of median value and the dotted line in the horizontal axis of box plot representing the mean fold of ABCB1 and ABCG2}.

Figure 2

Heat map analysis representing the normalised mRNA expression of ABCB1 and ABCG2 in tumour and normal tissue samples of all GBC patients [X axis represents the value of 2 ˆ ^ΔCt of ABCB1 and ABCG2 in tumour and normal samples, Y axis represents patients, positive value of Z scores represents higher expression and negative value represents lower expression].

Figure 3

Increased protein expression of ABCB1 (141 KDa) and ABCG2 (72 KDa) in tumour tissue of GBC patients. Beta actin (43 KDa) was used as loading control.

Figure 4

Kaplan–Meier Overall Survival graphs for various factors in GBC a) shows similar survival in male and female (P = 0.220), b) worse survival with poorly differentiated tumours although not statistically significant (P = 0.165) c-e) better survival in patients with papillary histology (P = 0.046), with T1-2 depth of invasion (P = 0.068), with absence of LN metastasis (P = 0.003) f) significantly better survival in early stages (P = 0.013), (g) significantly worse survival in patients with associated gallstone (P = 0.026) (h) no change in survival with or without chemotherapy (P = 0.71), (i–l) OS did not show any significant difference with change in expression of ABCB1 and ABCG2 at mRNA level (P = 0.672 and P = 0.941) and protein level (P = 0.509 and P = 0.467).

Figure 5

Kaplan–Meier Disease-Free Survival graphs for various factors in GBC (a) shows significantly better survival in male compared to female (P = 0.005), (b) significantly worse survival with poorly differentiated tumours (P = 0.048), (c–e) significantly better survival in patients with papillary histology (P = 0.010), with T1-2 depth of invasion (P = 0.016), with absence of LN metastasis (P = 0.002), (f) significantly better survival in early stages (P = 0.002), (h) significantly worse survival in patients with associated gallstone (P = 0.004), (g) no change in survival with or without chemotherapy (P = 0.40), (i–l) DFS did not show any significant difference with change in expression of ABCB1 and ABCG2 at mRNA level (P = 0.347 and P = 0.916) and protein level (P = 0.72 and P = 0.723).

Figure 6

Kaplan–Meier curves showing no difference in OS and DFS with change in expression of ABCB1 and ABCG2 at mRNA level (P = 0.804 and P = 0.101) and protein level (P = 0.839 and P = 0.176) in chemo group.

Figure 7

Kaplan–Meier curves showing no difference in OS and DFS with change in expression of ABCB1 and ABCG2 at mRNA level (P = 0.539 and P = 0.223) and protein level (P = 0.394 and P = 0.327) in non-chemo group.