Aldehyde dehydrogenase 1 (ALDH1) positivity correlates with poor prognosis in cervical cancer

Abstract

Objective

To analyse the associations between aldehyde dehydrogenase 1 (ALDH1) tumour immunopositivity and disease-free survival in cervical carcinoma.

Methods

ALDH1 immunohistochemistry was performed on formalin-fixed, paraffin wax-embedded cervical tumour tissue samples obtained from hospital archives. Data regarding disease-free survival were obtained. Kaplan–Meier survival analyses and Cox proportional hazards regression models were performed.

Results

Patients with ALDH1-positive tumours (n = 31) had significantly shorter disease-free survival than those with ALDH1-negative tumours (n = 167; 41.99 ± 0.90 vs 53.64 ± 2.67 months). ALDH1 positivity was associated with poor prognosis (relative risk 2.727; 95% confidence intervals 1.253, 5.914).

Conclusions

ALDH1 positivity is associated with poor prognosis of cervical carcinoma, and may be an independent predictor of prognosis.

Introduction

Cervical cancer is the third most common malignant tumour in Chinese women. ¹ The widespread use of cervical cytology screening has resulted in a substantial decrease in disease incidence and mortality, ² but the 5-year survival rate in patients with advanced disease is 3.2–13%. ³ Recurrence usually occurs within 2 years of diagnosis, ⁴ and the median survival time of recurring cases is only 7 months, due to resistance to traditional treatments. ⁵ Aldehyde dehydrogenase 1 (ALDH1) is a cancer stem-cell marker, the presence of which strongly correlates with tumour malignancy and self-renewal properties of stem cells in tumour types including breast cancer, hepatoma, lung and colon cancer.^6–9

Our previous study used immunohistochemistry to evaluated ALDH1 in 109 samples of cervical carcinoma and found that the presence of ALDH1 in tumour tissue was significantly related to lymph node metastasis and recurrence. ¹⁰ The aim of the present study was to analyse the relationship between ALDH1 positivity and cervical cancer prognosis.

Patients and methods

Tissue samples

Samples of formalin-fixed, paraffin wax-embedded cervical tumour tissue were obtained from the archives of the Department of Gynaecology, Sun Yat-sen Memorial Affiliated Hospital, Sun Yat-sen University, Guangzhou, China. Patients underwent treatment between October 2003 and December 2007 for confirmed cervical cancer (including squamous cell carcinoma and adenocarcinoma). Patients with evidence of disease on chest X-radiograph or magnetic resonance imaging (MRI) were excluded. Tumours were staged according to 2009 International Federation of Gynaecology and Obstetrics (FIGO) revised staging criteria. ¹¹ Data regarding treatment, myometrial invasion, vascular invasion and lymph node metastasis were obtained from medical records.

All patients provided written informed consent for the use of their tissue samples and the study was approved by the Ethics Committee of Sun Yat-sen Memorial Hospital, Sun Yat-sen University.

Follow up

Follow up continued until April 2009. Patients were followed up every 3 months in the first year, every 4 months in the second year, every 6 months for the next 3 years and annually thereafter. Each visit included a pelvic examination, cytology and colposcopy. Ultrasonography, MRI or computed tomography scan were performed if necessary.

Immunohistochemistry

Tissue was cut into 3-µm sections and mounted on glass slides pretreated with poly-l-lysine. Sections were boiled for 15 min in citrate buffer (10 mM, pH 6.0), cooled in the same buffer, then incubated for 10 min in 0.3% hydrogen peroxide to block endogenous peroxidase activity. Slides were incubated in mouse antihuman ALDH1 antibody (1 : 100 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4℃ in a humid chamber, and washed three times at room temperature in phosphate buffered saline (PBS; 0.01 M, pH 7.2; 3 min each wash). Sections were then incubated with horseradish peroxidase-conjugated antimouse secondary antibody (1 : 100 dilution; Zymed, San Francisco, CA, USA) for 60 min at room temperature, washed three times with PBS at room temperature (3 min each wash), then stained using the ABC Vectastain kit (Vector laboratories, Burlingame, CA, USA) according to the manufacturer’s instructions. All assays included positive and negative controls (replacement of primary antibody with mouse immunoglobulin G1 used as negative control). Immunohistochemical staining of ALDH1 was classified as negative (−, no staining), weakly positive (+, light-brown or yellow cells) or positive (++, brown staining). For the purpose of the study, ‘positive’ staining included both weakly positive and positive staining. All slides were evaluated by two independent pathologists.

Statistical analyses

Data were presented as mean ± SD. Kaplan–Meier analysis was used to estimate the probability of survival, and log-rank test was used to compare the survival differences. Potential prognostic factors (including age, pathological type, clinical stage, tumour differentiation, vessel invasion, deep myometrial invasion, lymph node metastasis, preoperative chemotherapy and ALDH1 immunopositivity) were analysed by multivariate Cox proportional hazards regression models and expressed as relative risk (RR) with 95% confidence intervals (CI). Statistical analyses were performed using SPSS® version 13.0 (SPSS Inc., Chicago, IL, USA) for Windows®. P-values <0.05 were considered statistically significant.

Results

The study included tumour tissue samples from 198 patients with cervical cancer (mean age 44.6 ± 8.7 years, age range 24–67 years). Patients were diagnosed with invasive squamous carcinoma (n = 155) or adenocarcinoma (n = 43). A total of 112 patients had undergone no preoperative radiotherapy or chemotherapy, four had undergone preoperative radiotherapy and 82 had received preoperative chemotherapy. Tumours were stage IB-1 (n = 53), stage IB-2 (n = 82), stage IIA (n = 49) and stage IIB (n = 14). A total of 28 patients were lost to follow-up (follow-up rate 85.9%). Mean follow-up duration was 35.13 ± 15.78 months (range 11–77 months).

Patients with ALDH1-positive tumours (n = 31) had significantly shorter disease-free survival than those whose tumours were ALDH1-negative (n = 167; 41.99 ± 0.90 vs 53.64 ± 2.67 months; P < 0.05; Figure 1).

Figure 1.

Kaplan–Meier analysis of disease-free survival in patients with cervical carcinoma, stratified according to the results of aldehyde dehydrogenase 1 (ALDH1) tumour immunohistochemistry (negative, n = 31; positive, n = 167). P < 0.05; log-rank test.

Lymph node metastasis (RR 5.445; 95% CI 2.014, 14.722) and ALDH1 positivity (RR 2.727; 95% CI 1.253, 5.914) were associated with poor prognosis.

Discussion

In the current study of well-characterized patients with cervical carcinoma and long-term follow-up, ALDH1 positivity was associated with poor disease-free survival. ALDH1 plays a role in the biological synthesis of retinoic acid and is involved in the early differentiation of stem cells by mediating the oxidation of retinol into retinoic acid. ¹² Retinoic acid is involved in cell differentiation and proliferation via binding to the retinoic acid receptor. ¹³

It is thought that cancer stem cells may be the original cells in tumour metastases. ¹⁴ ALDH1 is a marker for the identification of different types of cancer stem cell.^9,15,16 In bladder cancer, ALDH-1 levels are higher in lymph node-positive patients than in lymph node-negative patients, ¹⁷ suggesting that ALDH-1-positive cells promote invasion and metastasis. A clinical study indicated that breast cancer cells also express ALDH1. ¹⁸ The prognosis of patients with breast cancer who were ALDH1 positive was poor, and ALDH1 positivity was closely correlated with tumour grade, estrogen receptor/progestogen receptor status, overexpression of the oncogene ERBB2 and formation of cytokeratin.^6,19

In conclusion, ALDH1 positivity is associated with poor prognosis in cervical carcinoma and may be an independent predictor of prognosis.