Expression patterns of cancer stem cell markers ALDH1 and CD133 correlate with a high risk of malignant transformation of oral leukoplakia*

Authors

  • Wei Liu,

    1. Department of Oral Maxillofacial–Head and Neck Oncology, Shanghai Key Laboratory of Stomatology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People's Republic of China
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    • *

      W.L., L.W. and X.-M.S. contributed equally to this work.

  • Lan Wu,

    1. Department of Oral Mucosal Diseases, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People's Republic of China
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    • *

      W.L., L.W. and X.-M.S. contributed equally to this work.

  • Xue-Min Shen,

    1. Department of Oral Mucosal Diseases, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People's Republic of China
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    • *

      W.L., L.W. and X.-M.S. contributed equally to this work.

  • Lin-Jun Shi,

    1. Department of Oral Mucosal Diseases, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People's Republic of China
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  • Chen-Ping Zhang,

    1. Department of Oral Maxillofacial–Head and Neck Oncology, Shanghai Key Laboratory of Stomatology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People's Republic of China
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  • Li-Qun Xu,

    Corresponding author
    1. Department of Oral Maxillofacial–Head and Neck Oncology, Shanghai Key Laboratory of Stomatology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People's Republic of China
    • Shanghai Key Laboratory of Stomatology, 639 Zhizaoju Road, Shanghai 200011, People's Republic of China
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    • Tel.: +86-21-2327-1699, Fax: +86-21-6308-7076

  • Zeng-Tong Zhou

    Corresponding author
    1. Department of Oral Mucosal Diseases, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People's Republic of China
    • Shanghai Key Laboratory of Stomatology, 639 Zhizaoju Road, Shanghai 200011, People's Republic of China
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    • Tel.: +86-21-2327-1699, Fax: +86-21-6308-7076


Abstract

Molecular markers for predicting oral cancer development in premalignant oral leukoplakia (OL) are urgently needed. The objective of this study was to examine the expression patterns of cancer stem cell markers ALDH1 and CD133 in samples from patients with OL, and determine their prognostic values for subsequent development of oral cancer. Immunohistochemistry for ALDH1 and CD133 was performed in samples from a cohort of 141 patients with biopsy-proven OL who received a mean follow-up of 5.5 years. Patient clinicopathologic and follow-up data were analyzed. Expression of ALDH1 and CD133 was observed in 54 (38.3%) and 32 (22.7%) of 141 patients with OL, respectively. Kaplan–Meier analysis showed that 48.1% patients with ALDH1-positivity developed oral cancer compared with 12.6% those with ALDH1-negativity (p < 0.001). Meanwhile, 59.4% patients with CD133-positivity developed oral cancer compared with 16.5% those with CD133-negativity (p < 0.001). Multivariate analysis revealed that ALDH1 and CD133 expression was associated with 4.17-fold [95% confidence interval (CI), 1.96–8.90; p < 0.001] and 2.86-fold (95% CI, 1.48-5.55; p = 0.002) increased risk of OL transformation, respectively. Collectively, these data demonstrated for the first time that the expression of ALDH1 and CD133 correlated with malignant transformation in a large series of patients with OL who received a long-term follow-up, which suggests that they may serve as predictors to identify OL with a high risk of oral cancer development.

Head and neck squamous cell carcinoma (HNSCC), including oral squamous cell carcinoma (OSCC), is the sixth most common type of malignancy worldwide; early diagnosis of high-risk potentially malignant lesion is a high priority for reducing both morbidity and mortality.1–3 Increasing evidence indicates that the initiation, progression, recurrence and metastasis of HNSCC are related to the behavior of a small subpopulation of cancer stem cells (CSCs).4–6 Clinical and therapeutic implications of CSCs has attracted growing attention, including early detection and prognostication of cancer. In addition, CSCs can be identified and isolated by expression of distinctive markers to enrich for stem cells.7

Oral leukoplakia (OL) is the best-known potentially malignant disorder of OSCC, with a frequency of malignant transformation between 17% and 35%.8, 9 Histologic grading of epithelial dysplasia currently is the most important indicator for determining the risk of OL transformation; however, it is a well-known fact that this histologic classification is insufficient and may involve subjectivity.10 Furthermore, previous studies have demonstrated no evidence of an effective treatment to prevent malignant transformation; it is increasingly evident that the identification of high-risk OL before transformation is of utmost importance for effective intervention.11 Therefore, novel biomarkers are required to identify the OL lesions at a higher risk for transformation, which may provide opportunities for prophylactic intervention in the high-risk patient groups.

ALDH1 is the isoform of aldehyde dehydrogenase (ALDH) that expressed in humans is a cytosolic detoxifying isoenzyme that oxidizes intracellular aldehydes, and contributes to the oxidation of retinol to retinoic acid in early stem cell differentiation.12 Recent studies have shown that ALDH1 is a specific marker for identification of head and neck CSCs and plays a crucial role in maintaining the self-renewal properties and tumorigenicity in HNSCC-derived CSCs.13–16 Moreover, ALDH1 immunoexpression has been found in tissue sample of HNSCC,17, 18 and has prognostic value for HNSCC survival.18 Interestingly, Visus et al.19 observed that ALDH1 overexpression in samples of HNSCC and oral dysplasia, and suggested that it is a marker for distinguishing malignant from premalignant cells in HNSCC and is also an essential epitope for developing ALDH1-based vaccines for HNSCC therapy.

CD133, also known as Prominin-1 and AC133, was first described as a cell surface marker on hematopoietic stem cells and early progenitor cells in the bone marrow.20 Recent studies have shown that CD133 has been used as a marker to identify stem cells derived from primary solid cancers of lung and head and neck.21–24 Furthermore, CD133 immunoexpression has also been demonstrated to be a prognostic marker of OSCC survival.25 Recently, Ma et al.26 demonstrated that coexpression of ALDH1 and CD133 can more specifically characterize the tumorigenicity of liver CSC subpopulation. Jiang et al.27 observed that coexpression of ALDH1 and CD133 proteins in lung cancer specimens, indicating that both positive cells might be rich with lung CSCs.

In this study, we hypothesized that the expression patterns of ALDH1 and CD133, 2 stem cell markers of HNSCC, would be dysregulated in patients with OL. We thus examined the immunoexpression of ALDH1 and CD133 in samples from 141 patients with OL who received long-term follow-up, and determined their prognostic values for subsequent development of OSCC.

Abbreviations

ALDH: aldehyde dehydrogenase; CI: confidence interval; CSC: cancer stem cell; FFPE: formalin-fixed paraffin-embedded; HNSCC: Head and neck squamous cell carcinoma; HR: hazard ratio; OCFS: Oral cancer-free survival; OL: leukoplakia; OSCC: oral squamous cell carcinoma

Material and Methods

Patients and tissue specimens

A cohort of 218 patients with the clinical and pathologic diagnosis of OL between 1978 and 2008 was reviewed at the Department of Oral Mucosal Diseases, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine in our previous study.9 All the study patients underwent biopsy or surgery, and samples were obtained from formalin-fixed paraffin-embedded (FFPE) tissues. Because the paraffin blocks from baseline biopsies were unavailable before 1985, the 141 patients with OL between 1985 and 2010 were reviewed and retrieved in the same study institution. The cases of this study cohort were not the same as the cases of the previous cohort.9 For all the subjects, the treatments were grouped into medication (vitamin A/Chinese herb, n = 122) and surgery (n = 19). In our clinic, periodic follow-up examinations at intervals of every 6 months or fewer were recommended for patients with OL. The detailed inclusion criteria and pathologic examination of a new binary system of grading oral dysplasia of all the cases were previously described.9 In this study, latency of the progressor was defined as the interval from first biopsy of OL to subsequent OSCC development, and a minimum of 6 months of the latency was performed. Of 141 lesions, 76 (53.9%) were located at the tongue, followed by buccal mucosa (27.7%), gingiva (9.2%), palate (5.7%), and floor of mouth (3.5%). The sites of all lesions were classified as tongue and nontongue according to our report.9 In this retrospective follow-up study, malignant transformation versus nontransformation was considered as the surrogate for clinical outcome of patients with OL. Of the 141 patients with a mean follow-up of 5.5 years, 37 (26.2%) developed OSCC that was confirmed by histopathology at the same sites. This study was approved by our local Institutional Review Board.

Tissue processing and immunohistochemistry

Five-μm-thick serial tissue sections from FFPE tissue blocks of OL were mounted on positively charged glass slides. Immunohistochemical staining was done using the standard streptavidin-peroxidase method. The anti-ALDH1A1 rabbit monoclonal antibody (1:250 dilution, ab52492; Abcam, Cambridge, UK) and anti-CD133/1 mouse monoclonal antibody (1:50 dilution, AC133; Miltenyi, Bergisch Gladbach, Germany) was used to detect ALDH1 and CD133 expression, respectively. Cell membrane and/or cytoplasmic immunoreactivity in epithelium of OL was considered to indicate ALDH1 and CD133 positive expression. Sample evaluation of immunostaining was performed by oral pathologists (J.L. and L.Z.W.) blinded for the clinical data. To analyze the prognostic values for cancer development, immunoreactivity of ALDH1 and CD133 was classified into two categories. According to criteria of % cells staining described previously by Visus et al.,19 we classified staining in ≤5 and >5% of epithelial cells of OL as ALDH1 negative and positive, respectively. According to scoring of % cells staining described previously by Chiou et al.,25 we classified staining in ≤5 and ≥5% of epithelial cells of OL as CD133 negative and positive, respectively.

Statistics

Statistical analysis was carried out with the Fisher's exact test among qualitative variables and the Student's t-test among quantitative variables. Pearson correlation analysis was used to determine the relationship of ALDH1 and CD133 expression over all cases. Oral cancer-free survival (OCFS) was determined by using the Kaplan–Meier method and log-rank test for time to malignant event analysis. Cox regression model was applied to evaluate hazard ratio (HR) for OL malignant transformation. HR with 95% confidence interval (95% CI) and p values were reported. All tests were two-sided, and p values of <0.05 were accepted for statistical significance.

Results

Patient characteristics and ALDH1 and CD133 expression

In this retrospective follow-up study, the patients with OL were grouped as untransformed cases (n = 104) and malignant-transformed cases (n = 37). The characteristics and ALDH1 and CD133 expression patterns of these patients with OL is summarized in Table 1. A significant difference with regard to oral epithelial dysplasia (p = 0.041, Fisher's exact test) was noted, whereas differences in age, gender, lesion site, diet habit, smoking, alcohol intake and follow-up time were not found between the two groups. Representative histopathology of OL was showed in Supporting Information Figure S1.

Table 1. Characteristics and protein expression of patient with oral leukoplakia (OL)
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Expression patterns of ALDH1 and CD133 in serial tissue sample of OL were showed in Figure 1. Among the 141 cases, 54 (38.3%) and 32 (22.7%) showed positive expression of ALDH1 and CD133, respectively. We observed ALDH1 expression in 28 of 104 (26.9%) untransformed cases and in 26 of 37 (70.3%) malignant-transformed cases (p < 0.001, Fisher's exact test). Meanwhile, CD133 expression in 13 of 104 (12.5%) untransformed cases and in 19 of 37 (51.4%) malignant-transformed cases (p < 0.001, Fisher's exact test). In addition, significant differences in coexpression of both ALDH1 and CD133 (p < 0.001, Fisher's exact test) and expression of either ALDH1 or CD133 (p < 0.001, Fisher's exact test) were observed between the two groups. The positive correlation between ALDH1 and CD133 expression was significant (p = 0.001, Pearson correlation coefficient = 0.270).

Figure 1.

Expression patterns of ALDH1 and CD133 in serial tissue sample of the same patient with oral leukoplakia. (a) ALDH1 and CD133 negativity; (b) coexpression of both ALDH1 and CD133 positivity. Either expression patterns of (c) ALDH1 positivity and CD133 negativity and (d) ALDH1 negativity and CD133 positivity. Magnification, ×400. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

ALDH1 and CD133 expression and the risk of oral cancer

To investigate the time to malignant transformation of OL, the OCFS by Kaplan–Meier method using clinicopathologic factors and ALDH1 and CD133 expression was performed. In this series, ALDH1 and CD133 expression and grade of dysplasia were found to be the significant indicators using the log-rank test (Table 2). For all the 141 subjects, 11 of 87 (12.6%) ALDH1-negative cases and 26 of 54 (48.1%) ALDH1-positive cases developed into oral cancer, respectively (p < 0.001, log-rank test). Meanwhile, 18 of 109 (16.5%) CD133-negative cases and 19 of 32 (59.4%) CD133-positive cases developed into oral cancer, respectively (p < 0.001, log-rank test). Of interest, coexpression of both ALDH1 and CD133 (p < 0.001, log-rank test) and expression of either ALDH1 or CD133 (p < 0.001, log-rank test) in patients with OL were found to be strongly prognostic of malignant transformation (Fig. 2). In addition, 24/109 (22.0%) patients with high-grade dysplastic OL as compared to 13/32 (40.6%) those with low-grade dysplastic OL (p = 0.002, log-rank test) was noted.

Table 2. Kaplan-Meier and Cox regression analysis of oral cancer risk
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Figure 2.

Kaplan–Meier curve for OCFS rates by (a) ALDH1 negativity and positivity, (b) CD133 negativity and positivity, (c) both ALDH1 and CD133 positivity and (d) either ALDH1 or CD133 positivity.

To evaluate the oral cancer risk in patients with OL, clinicopathologic parameters and ALDH1 and CD133 expression were analyzed by the Cox proportional hazards model (Table 2). On univariate analysis, expression of ALDH1 (HR = 5.44; 95% CI, 2.60–11.37; p < 0.001) and CD133 (HR = 3.73; 95% CI, 1.95–7.14; p < 0.001) and grade of dysplasia (HR = 2.90; 95% CI, 1.45–5.81; p = 0.003) were significantly associated with increased risk of malignant transformation. Of interest, coexpression of both ALDH1 and CD133 (HR = 4.69; 95% CI, 3.28–12.86; p < 0.001) and expression of either ALDH1 or CD133 (HR = 5.42; 95% CI, 2.38-12.34; p < 0.001) were found to be strongly associated with increased risk of transformation.

To further assess the influence of each factor, multivariate analysis were performed to assess which factors remained independent indicators of transformation after adjusting for factors that were significant by univariate analysis. On multivariate analysis, the adjusted HR for transformation was 4.17 for ALDH1 expression (95% CI, 1.96–8.90; p < 0.001) and 2.86 for CD133 expression (95% CI, 1.48-5.55; p = 0.002), respectively. In addition, the prognostic of grade of dysplasia was still significant (adjusted HR = 2.36; 95% CI, 1.16-4.79; p = 0.018) but inferior to prognostic of these proteins expression.

Discussion

In our previous study,9 we evaluated the usefulness of a new binary system of grading dysplasia proposed by the World Health Organization (WHO) Collaborating Centre for Oral Cancer and Precancer in prediction the risk of malignant transformation of a large series of patients with OL in the study institution, and drew the conclusion that the binary histologic grading was a significant indicator for OL transformation. In this study, we are exploring molecular markers for predicting the risk of transformation of patients with OL in the same institution on the basis of CSC theory. A patch-field-carcinoma progression model of oral cancer proposed by Braakhuis et al.,28 hypothesized that oral cancer development started with a “patch stem cell” into an expanding subpopulation of stem cells escaped growth control, and eventually occurred event of transformation. ALDH1 and CD133 are two well-conducted studied CSC markers that have been implicated in several solid tumor types including oral cancer.4–7

To the best of our knowledge, this study determined for the first time that whether CSC markers ALDH1 and CD133 were the predictors of oral cancer risk in a large series of patients with OL who received a long-term follow-up. Kaplan–Meier analysis for 5-year OCFS rate showed that 41.3% of patients with ALDH1 positivity developed OSCC, compared with 8.9% of those with ALDH1 negativity; 52.0% of patients with CD133 positivity developed OSCC, compared with 12.5% of those with CD133 negativity. Multivariate analysis revealed that the expression of ALDH1 and CD133 was associated with 4.17-fold (p < 0.001) and 2.86-fold (p < 0.01) increased risk of transformation, respectively. Also, the prognostic values of both markers were superior to that of histologic grading associated with 2.36-fold (p > 0.01) increased risk.

ALDH1 as a CSC marker has been demonstrated to be upregulated in a subpopulation of cancer stem-like cell derived from HNSCC13–16. Chen et al.18 reported that immunoexpression of ALDH1 was strongly associated with worse survival prognosis of patients with HNSCC, and this study showed that it was strongly associated with worse malignant prognosis of patients with OL. Interestingly, we demonstrated that ALDH1 was expressed in 38.3% patients with OL, and Visus et al.19 reported that ALDH1 was expressed in 32.5% patients with oral dysplasia and 61.5% patients with HNSCC. There was similar expression pattern of ALDH1, and these may reflect a stepwise malignant transformation of OL.

CD133 as a CSC marker also has been demonstrated to be upregulated in a subset of cancer stem-like cell isolated from OSCC cells.21–24 Chiou et al.25 reported that immunoexpression of CD133 was associated with worse survival prognosis of patients with OSCC, and this study showed that CD133 also was associated with worse malignant prognosis of patients with OL. CD133 expressed in 22.7% patients with OL in this study, and it increasingly expressed in 53.8% patients with OSCC.25 Also, Zhang et al.22 observed that CD133-positive cancer stem-like cells in OSCC possess higher clonogenicity, invasiveness and increased in vivo tumorigenicity compared with CD133-negative counterparts.

Of interest, Sullivan et al.29 recently reported that ALDH1 but not CD133 protein expression was associated with poor survival of a large series of patients with primary non–small cell lung cancer. However, we observed that single marker or combined markers of ALDH1 and CD133 were associated with poor outcome of patients with OL developed oral cancer. It is plausible to speculate that there is cancer type specificity of CD133 in tumorigenesis and progression of various carcinomas. In addition, the significant correlation between ALDH1 and CD133 expression in OL was noted (p = 0.001), most likely due to the similar nature of CSC marker. Taken together, these data not only support the potential significance of ALDH1 and CD133 in early oral carcinogenesis but also suggest that both may be used as predictors for evaluating the risk of OL transformation, which were superior to the new binary system of grading dysplasia proposed by the WHO.

In this current follow-up cohort study, 26.2% patients with OL developed OSCC, within the range reported in the literature for OL.8 Herein, differences in treatments administered to the patients with OL were not observed (p > 0.05), and Cochrane Database of Systematic Review demonstrate no evidence of effective management in preventing the malignant transformation.11 This study showed that the grade of dysplasia was a significant indicator, but age, gender, oral site and oral habits were not significant risk factors of transformation of this cohort of patients with OL, in agreement with our previous study.9 In addition, earlier follow-up studies reported that immunoexpression of proteins, such as survivin, podoplanin, DeltaNp63, pChk2, DSPP and BSP, maybe predict the malignant risk.30 These biomarkers still require to be validated in further multicenter, longitudinal, prospective, large cohort studies. It is worth noting that the interpretation of protein expression of significant biomarkers is relatively simple in routine/diagnostic laboratories. Therefore, immunohistochemical staining of biomarkers is promising for the evaluation of the risk of oral cancer.

In summary, this study reported for the first time that the expression of ALDH1 and CD133, two CSC markers of HNSCC, was correlated with malignant transformation in a large series of patients with OL who received a long-term follow-up. Our findings support that a panel of CSC markers may serve as predictors for evaluating oral cancer risk of patients with OL, and patients with OL demonstrating positive ALDH1/CD133 expression should be followed carefully.

Acknowledgements

The authors thank Drs. Jiang Li and Li-Zhen Wang (Department of Oral Pathology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China) for technical support.

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