Neoadjuvant Chemotherapy Induces Expression Levels of Breast Cancer Resistance Protein That Predict Disease-Free Survival in Breast Cancer

<div><p>Three main xenobiotic efflux pumps have been implicated in modulating breast cancer chemotherapy responses. These are P-glycoprotein (Pgp), Multidrug Resistance-associated Protein 1 (MRP1), and Breast Cancer Resistance Protein (BCRP). We investigated expression of these proteins in breast cancers before and after neoadjuvant chemotherapy (NAC) to determine whether their levels define response to NAC or subsequent survival. Formalin-fixed paraffin-embedded tissues were collected representing matched pairs of core biopsy (pre-NAC) and surgical specimen (post-NAC) from 45 patients with invasive ductal carcinomas. NAC regimes were anthracyclines +/− taxanes. Immunohistochemistry was performed for Pgp, MRP1 and BCRP and expression was quantified objectively using computer-aided scoring. Pgp and MRP1 were significantly up-regulated after exposure to NAC (Wilcoxon signed-rank p = 0.0024 and p<0.0001), while BCRP showed more variation in response to NAC, with frequent up- (59% of cases) and down-regulation (41%) contributing to a lack of significant difference overall. Pre-NAC expression of all markers, and post-NAC expression of Pgp and MRP1 did not correlate with NAC response or with disease-free survival (DFS). Post-NAC expression of BCRP did not correlate with NAC response, but correlated significantly with DFS (Log rank p = 0.007), with longer DFS in patients with low post-NAC BCRP expression. In multivariate Cox regression analyses, post-NAC BCRP expression levels proved to predict DFS independently of standard prognostic factors, with high expression associated with a hazard ratio of 4.04 (95% confidence interval 1.3–12.2; p = 0.013). We conclude that NAC-induced expression levels of BCRP predict survival after NAC for breast cancer, while Pgp and MRP1 expression have little predictive value.</p></div

Representative staining patterns for Pgp, MRP1 and BCRP in matched breast tumour tissues pre- and post-neoadjuvant chemotherapy.

<p>Individual expression scores for each tissue shown, using a semi-automated scoring system, were: Pgp pre, 10.6; Pgp post, 117.9; MRP1 pre, 3.2; MRP1 post, 55; BCRP pre, 64.5; BCRP post, 89.7. Scale bar: 40 µm.</p

Post-NAC expression of BCRP predicts disease-free survival.

<p>Kaplan–Meier survival analyses for disease-free survival in patient groups with tumours with high or low post-NAC expression levels of Pgp (A), MRP1 (B) or BCRP (C). Cut-off used to dichotomise expression into low and high groups (Pgp: 90; MRP1∶21; BCRP: 47) were determined by ROC curve analyses (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062766#pone.0062766.s001" target="_blank">Fig. S1</a>).</p

Clinical and pathological features of the patients.

<p>Clinical and pathological features of the patients.</p

<i>In silico</i> analyses using the Oncomine platform showed that expression of the ESR2 gene in the breast tumour microenvironment does not correlate with patient outcome (p = 0.9337) (A). Expression of TGFBR2 (B) and BMPR2 (C) showed a trend towards significance (p = 0.1046 and p = 0.2029) suggesting that high expression levels of these genes are associated with lower rates of patient survival.

<p><i>In silico</i> analyses using the Oncomine platform showed that expression of the ESR2 gene in the breast tumour microenvironment does not correlate with patient outcome (p = 0.9337) (A). Expression of TGFBR2 (B) and BMPR2 (C) showed a trend towards significance (p = 0.1046 and p = 0.2029) suggesting that high expression levels of these genes are associated with lower rates of patient survival.</p

Laser micro-dissection (LMD) was used to isolate areas of epithelium in normal (A), DCIS (B) and invasive (C) breast tissue from the same tissue section. Breast tissue images show examples of the areas captured for patient 4 as a representative example. The expression of miR–92 decreased during breast cancer progression with highest levels observed in normal breast epithelium, decreasing in DCIS and being lowest in invasive breast tissue (D). *denotes significance of p<0.01.

<p>Laser micro-dissection (LMD) was used to isolate areas of epithelium in normal (A), DCIS (B) and invasive (C) breast tissue from the same tissue section. Breast tissue images show examples of the areas captured for patient 4 as a representative example. The expression of miR–92 decreased during breast cancer progression with highest levels observed in normal breast epithelium, decreasing in DCIS and being lowest in invasive breast tissue (D). *denotes significance of p<0.01.</p

Down-regulation of miR92 expression in normal fibroblasts (NFs), but not cancer associated fibroblasts (CAFs), significantly enhances the invasive capacity of breast cancer epithelial cells. Matched NFs and CAFs were reverse transfected with either an inhibitor of miR92 or negative control (A) and a Matrigel™ invasion assay was used to assess effects on the behaviour of breast cancer epithelial cells 48 hours post-transfection. Down-regulation of miR92 significantly increased the invasion of MCF7 (B) and MDA-MB–231 (C) cells. Error bars are ± S.D. *denotes significance of p<0.05; **denotes significance of p<0.01; ***denotes significance of p<0.001.

<p>Down-regulation of miR92 expression in normal fibroblasts (NFs), but not cancer associated fibroblasts (CAFs), significantly enhances the invasive capacity of breast cancer epithelial cells. Matched NFs and CAFs were reverse transfected with either an inhibitor of miR92 or negative control (A) and a Matrigel™ invasion assay was used to assess effects on the behaviour of breast cancer epithelial cells 48 hours post-transfection. Down-regulation of miR92 significantly increased the invasion of MCF7 (B) and MDA-MB–231 (C) cells. Error bars are ± S.D. *denotes significance of p<0.05; **denotes significance of p<0.01; ***denotes significance of p<0.001.</p

<i>In silico</i> analysis using BreastMark [26] shows luminal A breast cancer patients (n = 154) who express miR–92 have improved disease free survival (DFS) rate compared to patients with low expression levels with a hazard ratio of 0.49 (95% confidence intervals 0.28–0.84; p = 0.008).

<p><i>In silico</i> analysis using BreastMark [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139698#pone.0139698.ref026" target="_blank">26</a>] shows luminal A breast cancer patients (n = 154) who express miR–92 have improved disease free survival (DFS) rate compared to patients with low expression levels with a hazard ratio of 0.49 (95% confidence intervals 0.28–0.84; p = 0.008).</p

Expression of ERβ1 and miR–92 are not inversely correlated in patients with primary breast cancer. Staining patterns of ERβ1 showed nuclear expression in the normal breast (A) epithelium (examples shown by red arrows) with no significant difference in nuclear staining during cancer progression (B). A shift in the localisation of ERβ1 staining was observed with a significant increase in cytoplasmic staining (green arrows) during progression to DCIS (C, E) and invasive breast cancer (D, E). Blue arrows show ERβ1-negative nuclei. Images of breast tissues show the staining patterns for patient 4 as a representative example. Horizontal lines represent the mean. Range ± S.D. *denotes significance of p<0.05; **denotes significance of p<0.01.

<p>Expression of ERβ1 and miR–92 are not inversely correlated in patients with primary breast cancer. Staining patterns of ERβ1 showed nuclear expression in the normal breast (A) epithelium (examples shown by red arrows) with no significant difference in nuclear staining during cancer progression (B). A shift in the localisation of ERβ1 staining was observed with a significant increase in cytoplasmic staining (green arrows) during progression to DCIS (C, E) and invasive breast cancer (D, E). Blue arrows show ERβ1-negative nuclei. Images of breast tissues show the staining patterns for patient 4 as a representative example. Horizontal lines represent the mean. Range ± S.D. *denotes significance of p<0.05; **denotes significance of p<0.01.</p

Expression of miR92 in normal fibroblasts (NFs) and cancer associated fibroblasts (CAFs) in patient samples (A) and primary fibroblast cultures (B) by qRT-PCR. *denotes significance of p = 0.0014 between NFs (high and low expressing groups) and NFs and CAFs (ANOVA).

<p>Expression of miR92 in normal fibroblasts (NFs) and cancer associated fibroblasts (CAFs) in patient samples (A) and primary fibroblast cultures (B) by qRT-PCR. *denotes significance of p = 0.0014 between NFs (high and low expressing groups) and NFs and CAFs (ANOVA).</p