Changes in aldehyde dehydrogenase-1 expression during neoadjuvant chemotherapy predict outcome in locally advanced breast cancer

Abstract

Introduction: Although neoadjuvant chemotherapy (NAC) for locally advanced breast cancer can improve operability and local disease control, there is a lack of reliable biomarkers that predict response to chemotherapy or long-term survival. Since expression of aldehyde dehydrogenase-1 (ALDH1) is associated with the stem-like properties of self-renewal and innate chemoresistance in breast cancer, we asked whether expression in serial tumor samples treated with NAC could identify women more likely to benefit from this therapy.

Methods: Women with locally advanced breast cancer were randomly assigned to receive four cycles of anthracycline-based chemotherapy, followed by four cycles of taxane therapy (Arm A), or the same regimen in reverse order (Arm B). Tumor specimens were collected at baseline, after four cycles, and then at surgical resection. ALDH1 expression was determined by immunohistochemistry and correlated with tumor response using Fisher's exact test while Kaplan-Meier method was used to calculate survival.

Results: A hundred and nineteen women were enrolled into the study. Fifty seven (48%) were randomized to Arm A and 62 (52%) to Arm B. Most of the women (90%) had ductal carcinoma and 10% had lobular carcinoma. Of these, 26 (22%) achieved a pathological complete response (pCR) after NAC. There was no correlation between baseline ALDH1 expression and tumor grade, stage, hormone receptor, human epidermal growth factor receptor 2 (HER2) status and Ki67 index. ALDH1 negativity at baseline was significantly associated with pCR (P = 0.004). The presence of ALDH1(+) cells in the residual tumor cells in non-responding women was strongly predictive of worse overall survival (P = 0.024). Moreover, serial analysis of specimens from non-responders showed a marked increase in tumor-specific ALDH1 expression (P = 0.028). Overall, there was no survival difference according to the chemotherapy sequence. However, poorly responding tumours from women receiving docetaxel chemotherapy showed an unexpected significant increase in ALDH1 expression.

Conclusions: ALDH1 expression is a useful predictor of chemoresistance. The up-regulation of ALDH1 after NAC predicts poor survival in locally advanced breast cancer. Although the chemotherapy sequence had no effect on overall prognosis, our results suggest that anthracycline-based chemotherapy may be more effective at targeting ALDH1(+) breast cancer cells.

Trial registration: ACTRN12605000588695.

Figure 1

Study design. Flowchart depicting the examinations, imaging modalities and chemotherapy for women with locally advanced breast cancer.

Figure 2

Representative immunohistochemical staining intensity of ALDH1 for patients with breast cancer. (A) ALDH1 negative (0) result, (B) ALDH1 positive (1+), (C) = ALDH1 positive (2+) and (D) ALDH1 positive (3+). Strong staining was also observed on the normal ducts (E) and tumor stroma in some cases (F). Only cytoplasmic staining in the invasive tumor component was considered for final analysis. ALDH1, aldehyde dehydrogenase-1.

Figure 3

Overall survival analysis according to ALDH1 expression pre- and post-chemotherapy. The Kaplan-Meier curves illustrate the prognostic significance of ALDH1 pre and post neo-adjuvant chemotherapy (NAC) in patients with locally advanced breast cancer. (A) Before chemotherapy, there was no significant difference in overall survival in ALDH1(−) vs. ALDH1(+) groups. (B) At the end of chemotherapy, patients who were ALDH1(−) had much better overall survival (100% five-year survival) compared to those who were ALDH1(+) (66.5% five-year survival). (P = 0.045, HR 3.75, 95% CI 1.03 to 14.42). ALDH1, aldehyde dehydrogenase-1.

Figure 4

Prognostic significance of residual tumor at the end of NAC. These Kaplan-Meier curves depict that at the end of NAC, patients with complete pathological response (pCR) (blue line in A) had much better OS compared to those with a residual tumor at the end of NAC (green line in A). Surprisingly, patients with ALDH1(−) residual tumors at the end of NAC (purple line in A) also had much better OS. When combined into one group (purple line in B), patients with no residual tumor or ALDH1(−) residual tumor had significantly better OS compared to those with an ALDH1(+) residual tumor at the end of NAC (P = 0.005, HR 10.58, 95% CI 1.65 to 14.68). ALDH1, aldehyde dehydrogenase-1; NAC, neoadjuvant chemotherapy; OS, overall survival.

Figure 5

Illustration of chemoresistance in ALDH1(+) tumors. (A) Patients who did not have pCR to NAC, there was a statistically significant rise in median ALDH1 H-score at midpoint (after four cycles) as well as at the end (after eight cycles) of NAC. (B) Patients who achieved pCR could not be stained for ALDH1 due to lack of tumor cells at the end of eight cycles; however, after four cycles, there was no significant rise in median ALDH1 H-score. Horizontal bars indicate median H score (red) with 10^th to 90^th percentile (black). (_* = P-value <0.05, Kruskal Wallis test). ALDH1, aldehyde dehydrogenase-1; pCR, complete pathologic response; NAC, neoadjuvant chemotherapy.

Figure 6

Illustration of phenotypic switching in response to chemotherapy. Dynamic changes in the expression of ALDH1 were observed during NAC. Chart A illustrates that there was a switch from ALDH1(−) to ALDH1(+) phenotype (positive switch) in 27% of cases and from ALDH1(+) to ALDH1(−) phenotype (negative switch) in 19% of cases, after four cycles of chemotherapy. However, patients who remained ALDH1(−) both at baseline and after four cycles had the highest response rates, while those who were ALDH1(+) at both time points had the lowest response rates. Those who were ALDH1(+) at any time point had intermediate response rates (B). We observed a positive switch more often on patients receiving docetaxel (TAX) and a negative switch more often in patients receiving FEC chemotherapy (C). Finally, the overall survival of patients who remained ALDH1(−) throughout the eight cycles of NAC was significantly higher than those who were ALDH1(+) at any time point (D). (_* = P <0.05 Fishers exact test). ALDH1, aldehyde dehydrogenase-1; FEC, fluorouracil, epirubicin, cyclophosphamide; NAC, neoadjuvant chemotherapy.

Figure 7

The effect of chemotherapy type on ALDH1 expression. There was a significant rise in median ALDH1 H-score after docetaxel (TAX) therapy, whether received at the beginning (A) or after four cycles of FEC (B). Conversely, there was no significant change in the median H-score after FEC, whether received at the beginning or after four cycles of docetaxel (TAX). Horizontal bars indicate median H score (red) with 10^th to 90^th percentile (black). (_* = P-value <0.05, Mann–Whitney U test). ALDH1, aldehyde dehydrogenase-1; FEC, fluorouracil, epirubicin, cyclophosphamide.