Main

Aldehyde dehydrogenase (ALDH) is a family of ubiquitous enzymes located in nearly all mammalian tissues, which catalyze the oxidation of aldehydes to their carboxylic acid forms.1, 2 They participate in the detoxification of acetaldehyde,1, 2 the metabolism of biogenic amines,3 corticosteroids,4 and retinoic acid.5, 6

To date, 17 isoforms of ALDH had been described.7 ALDH1 is the cytosolic isoform. Murine and human hematopoietic stem/progenitor cells have been isolated based on their high levels of ALDH activity.8, 9, 10, 11, 12, 13 ALDH activity has been used to identify stem-like subsets not only in human hematopoietic cancers but also in solid cancers (eg, breast cancer).14, 15, 16, 17 A recent study by Ginestier et al15 demonstrated that high ALDH activity selected for both normal and tumorigenic human mammary epithelial cells with stem/progenitor properties, expression of ALDH1 was a predictor of poor clinical outcome in breast tumors. However, it remains controversial that these cell-surface markers can be used as the sole means to isolate cancer stem cells because of the heterogeneous nature of solid tumors.14, 18, 19, 20, 21, 22 Furthermore, the clinical, pathological, and immunohistochemical features of cancers associated with high expression of these markers remain unknown.

Ovarian cancer ranks as the fifth leading cause of cancer deaths among women.23 The expression of ALDH1 and its clinical significance is unknown in ovarian cancer. The authors hypothesized that the level of ALDH1 expression may be correlated with the clinical outcome of patients with ovarian cancer. The purpose of this study therefore was to evaluate the association between the expression of ALDH1 and the clinical pathological factors (including diagnosis, tumor grade, disease stage, and clinical response to chemotherapy), as well as the overall survival and disease-free survival of ovarian cancer. To do this, we retrospectively analyzed the clinical pathological factors of 442 cases of primary ovarian cancer and subjected archived tissue specimens to microarray analysis of the expression of putative cancer stem cell marker ALDH1 by immunohistochemical staining and correlate with the clinical outcome.

Materials and methods

Patients and Clinicopathologic Data

This study analyzed tumor samples from women diagnosed with primary ovarian carcinoma that had undergone initial surgery at The University of Texas MD Anderson Cancer Center between 1 January 1990 and 31 December 2005. Depending on the availability of representative tumor samples, we obtained data for 442 patients. The relevant clinical data were collected by retrospective review of the patients' files. Follow-up information was updated through May 2008 by reviewing medical records and the United States Social Security Index. Histopathologic diagnoses were based on World Health Organization criteria,24 tumor grading for non-serous carcinomas was based on Gynecologic Oncology Group criteria,25, 26, 27 and disease staging was assigned according to the International Federation of Gynecology and Obstetrics staging system.28 Serous carcinomas were graded by using a two-tier system (low grade and high grade) according to the criteria proposed by Malpica et al.29

To analyze response to primary therapy, we classified patients as responders or nonresponders. Patients who entered complete clinical remission with a normal CA125 level after chemotherapy for histological or cytological diagnosis of ovarian carcinoma with a treatment-free interval of ≥6 months were defined as responders.30

The group of nonresponders also was subdivided according to whether patients had progressive disease or recurrent disease. Progressive disease was defined as progression that occurred without disease remission observed after the initiation of treatment; recurrent disease was defined as disease that was detected after a period of clinically documented remission that was not sustained.31, 32

Construction of the Tissue Microarrays

We constructed tissue microarray blocks by taking one representative paraffin-embedded block from every patient and taking one core from morphologically representative areas of blocks as previously described.33 The use of tissue blocks and chart reviews was approved by the institutional review board of The University of Texas MD Anderson Cancer Center.

Immunohistochemical Analysis

Tissue microarray slides were subjected to immunohistochemical staining according to the manufacturer's protocol (Biocare Medical, Concord, CA, USA). In brief, after initial deparaffinization/hydration, sections were microwaved for 15 min in 10 mM citrate buffer, pH 6.0, to unmask the epitopes. Endogenous peroxidase activity was blocked by using 3% hydrogen peroxide. Nonspecific binding was blocked with background sniper (Biocare Medical) and slides were incubated for 10 min at room temperature. The slides were then incubated overnight at 4°C with primary mouse monoclonal antibody against ALDH1 (clone 44/ALDH, 1:100 dilution; BD Biosciences, San Jose, CA, USA); with a biotin-labeled secondary antibody (Universal Goat Link; Biocare Medical) for 15 min; and finally with HRP (Biocare Medical) for 15 min. Tissues were then stained for 5 min with 3,3′-diaminobenzidine (Biocare Medical). Finally, tissues were counterstained with hematoxylin, dehydrated, and mounted in DePex. Negative controls were made by replacing the primary antibody with phosphate-buffered saline. The intensely ALDH1-positive stromal cells were used as internal positive controls.

Immunohistochemical stainings for ALDH1 were analyzed by two gynecological pathologists (JL and BC). Staining scored only according to cytoplasmic staining of the cancer cells. The degree of staining was quantified using a four-score grading system. Cores with <5% ALDH1-positive cells were given a score of 0, those with 5–20% ALDH1-positive cells were given a score of 1, those with 20–50% ALDH1-positive cells were scored as 2, and those with >50% ALDH1-positive cells were scored as 3. For the statistical analysis, we divided cases into two groups: low expression (with scores of 0 or 1) and high expression (with scores of 2 or 3).

Statistical Analysis

Fisher's exact test and logistic regression analysis were performed to evaluate the association of ALDH1 with clinical factors. The Kaplan–Meier method was used to estimate the probability of overall survival and disease-free survival, and the log-rank test was used to compare the overall survival or disease-free survival between different comparison groups, such as patients with low or high ALDH1 expressions. Multivariate Cox proportional hazards regression models were fitted to determine the significant factors associated with overall survival and disease-free survival and to assess the association of ALDH1 with overall survival or disease-free survival after adjusting the effect of other clinical factors. The overall survival time was computed as the time interval from the date of first biopsy to the date of death or the last follow-up date, whichever occurred first. Patients alive on the last follow-up date were censored. The disease survival time was computed as the time period from the date of first biopsy to the date of recurrence, the date of death, or the date of last follow-up, whichever occurred first. Patients alive on the last follow-up date without recurrence were censored. Results were considered statistically significant at the P<0.05 level. SAS 9.1 software (SAS Institute Inc., Cary, NC, USA) was used for the statistical analysis.

Results

Patient Characteristics

The median age of the 442 patients was 60 years (range, 21–89 years). The median overall survival time was 4.0 years (95% CI: 3.5–4.6 years), and the overall survival rates were 60% (95% CI: 0.55–0.65) at 3 years, 42% (95% CI: 0.37–0.47) at 5 years, and 27% (0.22–0.32) at 10 years. The median follow-up interval was 8.0 years with a 95% confidence interval of 6.5–10.2 years.

ALDH1 Expression and Localization

Diffuse cytoplasmic staining with moderate intensity was observed in different proportion of the tumor cells. Very strong cytoplasmic and nuclear staining was observed in stromal cells. The expression in epithelial cancer cells was scored and subjected for statistical analysis. The percentage of positive cancer cells varied from <5 to >50% in our patient population (Figure 1).

Figure 1
figure 1

Immunoreactivity patterns of ALDH1 in ovarian serous adenocarcinomas (a–c) and endometrioid adenocarcinomas (d–f). (a) ALDH1-negative staining in serous carcinoma. (b) Serous carcinoma cells (≤20%) show cytoplasmic staining for ALDH1. (c) Diffuse positive staining for ALDH1 in serous carcinoma. (d) ALDH1-negative staining in endometrioid adenocarcinoma. (e) Endometrioid carcinoma cells (≤20%) show cytoplasmic staining for ALDH1. (f) Diffused positive staining for ALDH1 in endometrioid carcinoma (original magnification × 200).

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Association Between Expression of ALDH1 and Clinicopathologic Variables

The results of immunostaining of the tumor microarrays, organized according to clinicopathologic characteristics of the patients, are shown in Table 1. No ALDH1 was expressed (score=0) in 226 patients (51%), 1–5% of the cells expressed ALDH1 in 82 patients (19%; score also=0), 6–20% of the cells expressed ALDH1 in 49 patients (11%; score=1), 21–50% of the cells expressed ALDH1 in 46 patients (10%; score=2), and >50% of cells expressed ALDH1 in 41 patients (9%; score=3). High expression of ALDH1 (>20% of cells) was associated with endometrioid adenocarcinoma (P<0.0001), early-stage disease (P=0.006) (the cut-offs for early vs late stage is I vs II–IV), and low serum CA125 level (P=0.02). We analyzed the correlation between the expression of ALDH1 and the grade of serous carcinoma and endometrioid carcinoma separately because of different grading systems used in serous carcinoma and endometrioid carcinoma. No correlation was found between the expression of the ALDH1 and grade in either the serous or endometrioid carcinoma (data not shown).

Table 1 Correlations between the expression of ALDH and clinicopathologic factors
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The correlation of ALDH1 expression (high or low) with response to primary therapy is shown in Table 2. In total, 346 patients (78%) received postsurgical cisplatin-based treatment, either alone or in combination with other adjuvant drugs. In 46 patients (10%), cisplatin-based treatment was administered before surgical debulking surgery. Three patients (1%) received other forms of treatment (melphalan, 5-fluorouracil plus folinic acid). In 5 patients (1%), the treatment protocol was unknown. Overall, higher levels of expression of ALDH1 (>20%) were observed in the complete response group than in nonresponse group (P=0.0002). Similar proportions of ALDH1 expression were observed in both the postsurgical cisplatin-based treatment subgroup (P=0.01 and the presurgical cisplatin-based treatment subgroup (P=0.04).

Table 2 Correlation of ALDH1 expression and response to primary therapy
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The multivariate logistic regression analysis suggested that ALDH1 was significantly associated with histological type and response to chemotherapy (see Supplementary Table 1). Patients with endometrioid carcinoma would be more likely to have a higher ALDH1 expression (>20%) compared with those with serous adenocarcinoma (OR=6.12, P<0.0001). Patients with progression disease and recurrent disease would be less likely to have a higher ALDH1 expression (OR=0.31, 0.45 and P=0.03 and 0.03, respectively).

Association of ALDH1 with Overall Survival and Disease-Free Survival

Overall survival and disease-free survival rates at 3, 5, and 10 years are shown in relation to the expression of ALDH1 in Tables 3 and 4. At the time of this report, 88 of the 442 analyzed patients were alive without clinical evidence of ovarian carcinoma, 75 were alive with ovarian carcinoma, 262 had died of ovarian carcinoma, 15 were alive with unknown ovarian carcinoma status, and 2 had been lost to follow-up who were excluded from the overall survival and disease-free survival analysis. ALDH1 expression correlated with overall survival as well as disease-free survival. Patients who had tumors with >20% ALDH1-positive cells had better overall survival rate (P=0.01) and disease-free survival rate (P=0.006) than patients who had tumors with <20% ALDH1-positive cells (Figure 2).

Table 3 ALDH1 expression and overall survival
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Table 4 ALDH1 expression and disease-free survival
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Figure 2
figure 2

Kaplan–Meier survival curves for groups of ovarian carcinoma patients with low and high levels of ALDH1 expression. (a) Overall survival curves in all patients with ovarian cancer (n=440). (b) Disease-free survival curves in all patients (n=440).

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The multivariate Cox proportional hazards regression analysis indicated that stage was significantly associated with overall survival and disease-free survival. Patients with stage II, III, and IV would have a higher risk to be dead compared to stage I patients (HR=4.32, 7.2, 9.6 and P=0.01, 0.0002, 0.00002, respectively). Patients with stage II, III, and IV would have a higher risk of event (ie, recurrence, progression, or death) compared to stage I patients (HR=2.4, 5.7, 7.3 and P=0.048, <0.0001, <0.0001, respectively).

Discussion

In the current study of 442 well-characterized patients with long-term follow-up, high levels of ALDH1 expression were observed in 19% of the ovarian carcinoma samples, which correlated with endometrioid adenocarcinoma, early- stage disease, complete response to chemotherapy, and low serum CA125 level and favorable survival. In our study, multivariate Cox proportional hazards regression analysis showed that the early stage of disease was strongly associated with longer overall survival and disease-free survival (P<0.0002 and P<0.0001, respectively) and was a independent prognosis predicator. Although ALDH1 was not an independent predictor in multivariate analysis, high expression of ALDH1 was associated with early-stage disease (P=0.006). It gave us a clue that ALDH1 might be a potential-independent prognosis predictor in ovarian cancer. Our results are contrast with the report by Ginestier et al15 who demonstrated that the expression of ALDH1 was an independent predictor of poor clinical outcome in breast tumors. We found that high expression of ALDH1 was associated longer overall survival in patients with ovarian carcinoma. ALDH1 may have a different function in ovarian cancer than it does in breast cancer.

ALDH1 has been demonstrated to be a stem cell marker in several types of malignancies.14, 15, 16, 17 Theoretically, a high proportion of cancer stem cells in the tumor should be correlated with a poor prognosis. However, depending on the cancer site, markers used to identify stem cells from one organ may or may not be useful for identifying stem cells from other organs or tumor types.18, 34, 35 Our result, which is based on a large body of clinical material, demonstrates that expression of ALDH1 is correlated with favorable clinical outcome in patients with ovarian carcinoma. It remains to be determined whether ALDH1 is associated with a stem cell or stem-like cells in human ovarian cancer.

In summary, the current results demonstrate that in contrast to its function in breast cancer, ALDH1 has a favorable function in ovarian carcinoma and thus high expression of ALDH1 is a favorable prognostic factor in patients with ovarian cancer. ALDH1 thus may have a different function in ovarian cancer than it does in breast cancer.