ALDH-1 expression levels predict response or resistance to preoperative chemoradiation in resectable esophageal cancer patients

Abstract

Purpose: Operable thoracic esophageal/gastroesophageal junction carcinoma (EC) is often treated with chemoradiation and surgery but tumor responses are unpredictable and heterogeneous. We hypothesized that aldehyde dehydrogenase-1 (ALDH-1) could be associated with response.

Methods: The labeling indices (LIs) of ALDH-1 by immunohistochemistry in untreated tumor specimens were established in EC patients who had chemoradiation and surgery. Univariate logistic regression and 3-fold cross validation were carried out for the training (67% of patients) and validation (33%) sets. Non-clinical experiments in EC cells were performed to generate complimentary data.

Results: Of 167 EC patients analyzed, 40 (24%) had a pathologic complete response (pathCR) and 27 (16%) had an extremely resistant (exCRTR) cancer. The median ALDH-1 LI was 0.2 (range, 0.01-0.85). There was a significant association between pathCR and low ALDH-1 LI (p ≤ 0.001; odds-ratio [OR] = 0.432). The 3-fold cross validation led to a concordance index (C-index) of 0.798 for the fitted model. There was a significant association between exCRTR and high ALDH-1 LI (p ≤ 0.001; OR = 3.782). The 3-fold cross validation led to the C-index of 0.960 for the fitted model. In several cell lines, higher ALDH-1 LIs correlated with resistant/aggressive phenotype. Cells with induced chemotherapy resistance upregulated ALDH-1 and resistance conferring genes (SOX9 and YAP1). Sorted ALDH-1+ cells were more resistant and had an aggressive phenotype in tumor spheres than ALDH-1- cells.

Conclusions: Our clinical and non-clinical data demonstrate that ALDH-1 LIs are predictive of response to therapy and further research could lead to individualized therapeutic strategies and novel therapeutic targets for EC patients.

Keywords: ALDH-1; Chemotherapy resistance; Esophageal carcinoma; Predictive markers; Prognosis; Radiation resistance.

Figure 1

Immunohistochemistry staining of ALDH‐1 in untreated EC tumor of a patient with pathCR showing low LI (upper row) and a patient with exCRTR showing high LI (lower row). Abbreviations: pathCR, pathologic complete response; exCRTR, extreme resistance; and LI, labeling index.

Figure 2

Area under the receiver operating characteristic curve for pathCR (pathologic complete response) with C‐index of 0.798).

Figure 3

Area under the receiver operating characteristic curve for exCRTR (pathologic extreme resistance) with C‐index of 0.960).

Figure 4

EC cells with high ALDH‐1 LIs are resistant to chemotherapy and upregulate resistance‐conferring genes. A. ALDH1 activity assessed by fluorescence activated cell sorting in 3 cell lines (FLO‐1, JHESO, and OAPC) according to ALDEFLUOR detection kit following the protocol and diethylaminobenzaldehyde (DEAB) was used to inhibit ALDH‐1 activity to document the detection specificity. B. Bar graphs demonstrating the percentage of ALDH‐1 positive cells in 3 cell lines. C. Immunoblotting of 3 cell lines demonstrates upregulation of resistance conferring CSC genes are more upregulated in cells with higher percentage of ALDH‐1. D. 3 cell lines exposed to various concentrations of 5‐FU and the degree of response (cells with high percentage of ALDH‐1 are more resistant).

Figure 5

Cells with higher percentage of ALDH‐1 more readily for tumor sphere and are resistant to 5‐FU. A. From JHESO, ALDH‐1+ and ALDH‐1− cells are sorted by fluorescence activated cell sorting in 3 cell lines (FLO‐1, JHESO, and OAPC) according to ALDEFLUOR detection kit following the protocol and tumor sphere assays were carried out in triplicate in ultra‐low attachment plate in the tumor sphere medium. After 8–10 days in culture, the number and size of the tumor spheres were counted under a microscope. The ALDH‐1+ cells formed larger tumor spheres and these are numerically shown in the bar graph below. B. The proliferation rate of ALDH1+ and ALDH‐1− cells using the MTS proliferation assay on day 2 and day 6 demonstrate higher proliferative activity of ALDH‐1+ cells. C. ALDH‐1+ cells are more resistant to 5‐FU at different concentrations.