- Email: [email protected]
Bmi1 expression in oral lichen planus and the risk of progression to oral squamous cell carcinoma
Annals of Diagnostic Pathology xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Annals of Diagnostic Pathology
Original Contributions
Bmi1 expression in oral lichen planus and the risk of progression to oral squamous cell carcinoma Lihua Ma DDS a, 1,⁎, Hao Wang MD b, 1, Hui Yao DDS c, Laikuan Zhu DDS c, Wei Liu MD c,⁎, Zengtong Zhou DDS c a b c
School of Stomatology, Zhengzhou University, Zhengzhou, Henan, China Department of Urology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China Shanghai Key Laboratory of Stomatology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
a r t i c l e
i n f o
Available online xxxx Keywords: Bmi1 Cancer stem cell Oral lichen planus Malignant progression Oral squamous cell carcinoma
a b s t r a c t Oral lichen planus (OLP) is a potentially malignant disorder associated with an increased risk of progression to oral squamous cell carcinoma (OSCC). The objective of this study was to determine protein expression of cancer stem cell factor Bmi1 in a longitudinal series of patients with OLP and evaluate the correlation between Bmi1 expression and the risk of progression to OSCC. In a retrospective study, Bmi1 expression was determined using immunohistochemistry in samples from 96 patients with OLP who received a mean follow-up of 54 months, including patients who did not progress to OSCC (n = 87) and patients who had progressed to OSCC (n = 9). Analysis of 10 cases of normal oral mucosa and 6 cases of postmalignant OSCC form previously diagnosed OLP was also performed. The results showed that Bmi1 expression was observed in 32 (36.8%) of 87 cases of nonprogressing OLP and in 8 (88.9%) of 9 cases of progressing OLP. Bmi1 was not expressed in normal oral mucosa, but it was positively expressed in the 6 (100%) cases of OSCC. Multivariate analysis revealed that the risk of malignant progression in the patients with Bmi1-positive expression was significantly higher than those with Bmi1 negativity (odds ratio, 20.75; 95% confidence interval, 2.21-194.57; P = .008). Collectively, Bmi1 expression was significantly associated with malignant transformation in a large series of patients with OLP who received a longitudinal observation. Our findings suggested that Bmi1 may serve as a useful marker for the identification of a high risk of malignant progression of OLP. Crown Copyright © 2013 Published by Elsevier Inc. All rights reserved.
1. Introduction Oral lichen planus (OLP) is a relatively common inflammatory mucocutaneous disease of unknown etiology. It has a protracted clinical course despite various available treatments [1,2]. Patients with OLP carry a significantly increased risk of developing oral squamous cell carcinomas (OSCCs) [3]. Development of OSCC could arise from all clinical types of OLP, with a rate of malignant progression up to 6.4% [4]. Oral lichen planus has been defined as one of potentially malignant disorders by the World Health Organization [5], but clinical and histologic features of OLP lesions have limited prognostic value for malignant progression. Previous cross-sectional studies reported that desmocollin-1, matrix metallopeptidase, Smad, p53, and p16 may be relevant to malignant progression of OLP [6–10]. Obviously, the longitudinal observation
⁎ Corresponding authors. Tel.: +86 21 23271699; fax: +86 21 63087076. E-mail addresses: [email protected] (L. Ma), [email protected] (W. Liu). 1 L. Ma and H. Wang contributed equally to this work.
study on molecular event of the progression is of more scientific and clinical significance [11]. Bmi1 is a polycomb group transcription repressor that mediates gene silencing by regulating chromatin structure. It is considered to be a stem cell–related gene that has been demonstrated to play a role in the self-renewal of cancer stem cells [12]. Bmi1 has been demonstrated to be up-regulated in a subpopulation of cancer stemlike cells isolated from head and neck cancer cells [13–15]. Furthermore, it has been demonstrated to be a prognostic marker in several human cancers including gastric cancer and head and neck cancer [16–18]. Bmi1 was also found to be overexpressed in human gastrointestinal and oral dysplasias, indicating that its abnormal expression occurs early in gastrointestinal and oral carcinogenesis [19,20]. In the current study, we hypothesized that expression of Bmi1 protein would be dysregulated in patients with OLP. In this retrospective follow-up study, we examined protein expression of Bmi1 in samples of nonprogressing OLP, progressing OLP to postmalignant OSCC, and determined the correlation between Bmi1 expression in OLP and the risk of OSCC.
1092-9134/$ – see front matter. Crown Copyright © 2013 Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.anndiagpath.2013.03.002
Please cite this article as: Ma L, et al, Bmi1 expression in oral lichen planus and the risk of progression to oral squamous cell carcinoma, Ann Diagn Pathol (2013), http://dx.doi.org/10.1016/j.anndiagpath.2013.03.002
2
L. Ma et al. / Annals of Diagnostic Pathology xxx (2013) xxx–xxx
Table 1 Characteristics of patients with OLP Characteristic, n (%)
Nonprogressing OLP
Progressing OLP
Patients Follow-up (mo) Mean Range Age (y) Mean (SD) Range Sex Female Male Smoking history Never Past and present Unknown Alcohol intake Never Past and present Unknown Bmi1 expression Negative Positive
87 Time 1 53 16-175
9 Time 2 55 10-132
46.4 (11.7) 26-74
54.9 (8.9) 41-70
66 (75.9) 21 (24.1)
8 (88.9) 1 (11.1)
65 (81.2) 15 (18.8) 7 (−)
6 (75.0) 2 (25.0) 1 (−)
61 (77.2) 18 (22.8) 8 (−)
6 (75.0) 2 (25.0) 1 (−)
55 (63.2) 32 (36.8)
1 (11.1) 8 (88.9)
P .808
.038
.341
.648
1.00
1990 to 2010. Information regarding age, sex, smoking, and alcohol use at the time of the initial diagnosis of OLP was documented. All the study patients underwent biopsy. The biopsy was fixed in formalin, embedded in paraffin, and processed for routine histopathologic examination. The modified World Health Organization criteria for OLP [21] were used when examining the histopathology of the sections. In our clinic, periodic follow-up examinations at intervals of every 6 months or less were recommended for patients with OLP. Patient with nonprogressing OLP (n = 87) was defined as the one who did not progress to OSCC in our follow-up period, and patient with progressing OLP (n = 9) was defined as that subsequently progressed to OSCC confirmed by histopathology at the same oral site of OLP lesion in the follow-up. Ten samples of normal oral mucosa identified in biopsies taken for unrelated benign lesions were used as controls. Six samples of paraffin blocks of postmalignant OSCC lesions from previously diagnosed OLP were also studied. This study was approved by the local institutional review board.
.004
Time 1: From the initial diagnosis of OLP to the last consultation. Time 2: From the initial diagnosis of OLP to progress to carcinoma.
2. Materials and methods 2.1. Patients and tissue specimens A series of 96 patients with the clinical and pathologic diagnosis of OLP in this study were retrieved among the archived files at the Department of Oral Mucosal Diseases, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, from
2.2. Tissue processing and immunohistochemistry Four-micrometer-thick tissue sections from formalin-fixed, paraffin-embedded tissue blocks of the tissue samples were mounted on positively charged glass slides. Immunohistochemical staining was performed using Leica Automatic Stainer (Leica Microsystems Ltd, Wetzlar, Germany) following manufacturer's protocol. The anti-Bmi1 monoclonal antibody (1:150 dilution, ab14389; Abcam, Cambridge, UK) was used to detect Bmi1 expression. In negative controls, the primary antibody was replaced by nonimmune immunoglobulin G of the same isotype to ensure specificity. Cell nuclear immunoreactivity in the epithelium was considered to indicate positive expression of Bmi1. Sample evaluation of immunostaining was performed by oral pathologists (JL and LZW) blinded for the clinical data. According to
Figure. Representative expression pattern of Bmi1. Bmi1-negative expression in normal oral mucosa (A) and nonprogressing OLP (B). Magnification, ×200. Bmi1-positive expression in progressing OLP (C) and post-malignant OSCC from OLP (D). Magnification, ×400.
Please cite this article as: Ma L, et al, Bmi1 expression in oral lichen planus and the risk of progression to oral squamous cell carcinoma, Ann Diagn Pathol (2013), http://dx.doi.org/10.1016/j.anndiagpath.2013.03.002
L. Ma et al. / Annals of Diagnostic Pathology xxx (2013) xxx–xxx
3
4. Discussion
Table 2 Logistic regression analysis of the risk of oral cancer
In this retrospective follow-up study, the 96 patients with OLP were grouped as nonprogressing OLP (n = 87) and progressing OLP (n = 9). The baseline characteristics of these patients are presented in Table 1. A significant difference with regard to age (P = .038, Student t test) was found, whereas differences in follow-up period, sex, smoking history, and alcohol intake were not observed between the 2 groups. The representative immunoexpression of Bmi1 in normal oral mucosa, nonprogressing OLP, progressing OLP, and OSCC form previously diagnosed OLP were shown in the Figure. We observed that Bmi1-positive expression was absent in 10 samples of normal oral mucosa, but it expressed in the 6 cases (100%) of OSCC from OLP. Bmi1 expression was found in 32 (36.8%) of 87 cases of nonprogressing OLP and in 8 (88.9%) of 9 cases of progressing OLP (P = .004, Fisher exact test). Bmi1 expression was significantly increased from normal oral mucosa (0%), nonprogressing OLP (36.8%), progressing OLP (88.9%) to OSCC (100%) (P b .001, Fisher exact test).
Increasing evidence indicates that the initiation, progression, recurrence, and metastasis of head and neck squamous cell carcinoma are related to the behavior of a small subpopulation of cancer stem cells [22–24]. Clinical and therapeutic implications of cancer stem cells have attracted growing attention, including early detection and prognostication of cancer. In addition, cancer stem cells can be identified and isolated by expression of distinctive markers to enrich for stem cells [22–24]. Bmi1 is one of the well-conducted studied cancer stem cells factors, which has been implicated in several solid tumor types including head and neck squamous cell carcinoma [12]. Bmi1 has been studied as an oncoprotein associated with poor prognosis in several human cancers [16–18]. Recent studies have reported that Bmi1, as one of the stem cell markers, was essential for maintaining the self-renewal properties of and tumor progression in head and neck cancer [25–27]. Elevated Bmi-1 expression was demonstrated to be associated with dysplastic cell transformation during oral carcinogenesis and to be required for cancer cell replication and survival [20]. In the previous study, we reported Bmi1 abnormal expression in samples of oral leukoplakia [28] and suggested that it is a predictor for transformation and tumorigenicity of OSCC. In the current follow-up study, we attempted to determine Bmi1 expression pattern in samples of a series of patients with OLP and analyze the correlation between it expression and malignant potential of OLP. One unique feature of the current study is the 2-stage experimental design, which incorporated testing and validation steps. Oral carcinogenesis is a multistep process of cumulative genetic and epigenetic aberrations. First, we observed that Bmi1 expression was significantly increased from normal oral mucosa, nonprogressing OLP, progressing OLP to OSCC. Bmi1 expression level was consistent with malignant potential of oral mucosa, which may reflect a stepwise transformation of OLP into OSCC. Second, we focused on differential expression of Bmi1 in nonprogressing OLP and progressing OLP and association with the risk of malignant progression. The results showed that Bmi1 expression is of significant difference between the 2 groups, and the risk of malignant progression in the patients with Bmi1positive expression was significantly higher than those with Bmi1negative expression. Collectively, these data not only support that the Bmi1 might play a critical role in transformation and tumorigenicity of OSCC but also suggest that it was a potential biomarker for early detection of OLP malignant progression, although the biological function of Bmi1 as a putative cancer stem cell marker in oral potentially malignant lesions remains unclear. In summary, Bmi1 expression was significantly associated with malignant progression in a series of patients with OLP who received a long-term follow-up. Our findings suggested that Bmi1 may serve as a useful marker for the identification of a high risk of oral potentially malignant lesions progressing to OSCC.
3.2. Bmi1 expression in OLP and risk of OSCC
Acknowledgments
To evaluate the risk of OSCC in patients with OLP, Bmi1 expression and clinical characteristics of the patients in the current study were analyzed by logistic regression (Table 2). In the univariate analysis, age (OR, 1.07; 95% CI, 1.00-1.13; P = .047) and Bmi1 expression (OR, 13.75; 95% CI, 1.64-115.02; P = .016) were significantly associated with increased risk of malignant transformation of OLP. To further assess the influence of patient' age, we then did a multivariate analysis. After adjusting for age, Bmi1 expression retained statistical significance. The risk of malignant progression in the patients with Bmi1-positive expression was significantly higher than those with Bmi1-negative expression (adjusted OR, 20.75; 95% CI, 2.21-194.57; P = .008).
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. This study was supported by Shanghai Health Bureau (ZYSNXD-CC-ZDYJ004) and Science and Technology Commission of Shanghai (11DZ1972600).
Characteristic Univariate analysis Age Sex Smoking Alcohol intake Bmi1 expression Multivariate analysis Age Bmi1 expression
OR (95% CI)
P
1.07 (1.00-1.13) 0.39 (0.05-3.33) 1.44 (0.27-7.88) 1.13 (0.21-6.09) 13.75 (1.64-115.02)
.047 .391 .671 .887 .016
1.09 (1.01-1.17) 20.75 (2.21-194.57)
.021 .008
criteria of staining intensity described previously by Kang et al [20], we classified negative or barely detectable epithelium as Bmi1negative expression and weak, moderate to strong staining of epithelium as Bmi1-positive expression.
2.3. Statistics Statistical analysis was carried out with the χ 2 test or Fisher exact test among qualitative variables and the Student t test among quantitative variables. Logistic regression was applied to evaluate relative risk for OLP malignant transformation. Odds ratio (OR) with 95% confidence interval (95% CI) and P values were reported. All tests were 2 sided, and P b .05 was accepted for statistical significance.
3. Results 3.1. Patient characteristics and Bmi1 expression
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Please cite this article as: Ma L, et al, Bmi1 expression in oral lichen planus and the risk of progression to oral squamous cell carcinoma, Ann Diagn Pathol (2013), http://dx.doi.org/10.1016/j.anndiagpath.2013.03.002
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[3] Warnakulasuriya S, Kovacevic T, Madden P, et al. Factors predicting malignant transformation in oral potentially malignant disorders among patients accrued over a 10-year period in South East England. J Oral Pathol Med 2011;40:677–83. [4] Gonzalez-Moles MA, Scully C, Gil-Montoya JA. Oral lichen planus: controversies surrounding malignant transformation. Oral Dis 2008;14:229–43. [5] Sarode SC, Sarode GS, Karmarkar S, et al. A new classification for potentially malignant disorders of the oral cavity. Oral Oncol 2011;47:920–1. [6] Mattila R, Alanen K, Syrjänen S. Desmocollin expression in oral atrophic lichen planus correlates with clinical behavior and DNA content. J Cutan Pathol 2008;35: 832–8. [7] Chen Y, Zhang W, Geng N, et al. MMPs, TIMP-2, and TGF-beta1 in the cancerization of oral lichen planus. Head Neck 2008;30:1237–45. [8] Danielsson K, Wahlin YB, Coates PJ, et al. Increased expression of Smad proteins, and in particular Smad3, in oral lichen planus compared to normal oral mucosa. J Oral Pathol Med 2010;39:639–44. [9] Ebrahimi M, Nylander K, van der Waal I. Oral lichen planus and the p53 family: what do we know? J Oral Pathol Med 2011;40:281–5. [10] Poomsawat S, Buajeeb W, Khovidhunkit SO, Punyasingh J. Overexpression of cdk4 and p16 in oral lichen planus supports the concept of premalignancy. J Oral Pathol Med 2011;40:294–9. [11] Gonzalez-Moles MA, Scully C, Ruiz-Avila I. Molecular findings in oral premalignant fields: update on their diagnostic and clinical implications. Oral Dis 2012;18:40–7. [12] Siddique HR, Saleem M. Role of BMI1, a stem cell factor, in cancer recurrence and chemoresistance: preclinical and clinical evidences. Stem Cells 2012;30:372–8. [13] Prince ME, Sivanandan R, Kaczorowski A, et al. Identification of a subpopulation of cells with cancer stem cell properties in head and neck squamous cell carcinoma. Proc Natl Acad Sci USA 2007;104:973–8. [14] Zhang P, Zhang Y, Mao L, et al. Side population in oral squamous cell carcinoma possesses tumor stem cell phenotypes. Cancer Lett 2009;277:227–34. [15] Yanamoto S, Kawasaki G, Yamada S, et al. Isolation and characterization of cancer stemlike side population cells in human oral cancer cells. Oral Oncol 2011;47:855–60. [16] Liu JH, Song LB, Zhang X, et al. Bmi-1 expression predicts prognosis for patients with gastric carcinoma. J Surg Oncol 2008;97:267–72.
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Please cite this article as: Ma L, et al, Bmi1 expression in oral lichen planus and the risk of progression to oral squamous cell carcinoma, Ann Diagn Pathol (2013), http://dx.doi.org/10.1016/j.anndiagpath.2013.03.002
Contents lists available at SciVerse ScienceDirect
Annals of Diagnostic Pathology
Original Contributions
Bmi1 expression in oral lichen planus and the risk of progression to oral squamous cell carcinoma Lihua Ma DDS a, 1,⁎, Hao Wang MD b, 1, Hui Yao DDS c, Laikuan Zhu DDS c, Wei Liu MD c,⁎, Zengtong Zhou DDS c a b c
School of Stomatology, Zhengzhou University, Zhengzhou, Henan, China Department of Urology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China Shanghai Key Laboratory of Stomatology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
a r t i c l e
i n f o
Available online xxxx Keywords: Bmi1 Cancer stem cell Oral lichen planus Malignant progression Oral squamous cell carcinoma
a b s t r a c t Oral lichen planus (OLP) is a potentially malignant disorder associated with an increased risk of progression to oral squamous cell carcinoma (OSCC). The objective of this study was to determine protein expression of cancer stem cell factor Bmi1 in a longitudinal series of patients with OLP and evaluate the correlation between Bmi1 expression and the risk of progression to OSCC. In a retrospective study, Bmi1 expression was determined using immunohistochemistry in samples from 96 patients with OLP who received a mean follow-up of 54 months, including patients who did not progress to OSCC (n = 87) and patients who had progressed to OSCC (n = 9). Analysis of 10 cases of normal oral mucosa and 6 cases of postmalignant OSCC form previously diagnosed OLP was also performed. The results showed that Bmi1 expression was observed in 32 (36.8%) of 87 cases of nonprogressing OLP and in 8 (88.9%) of 9 cases of progressing OLP. Bmi1 was not expressed in normal oral mucosa, but it was positively expressed in the 6 (100%) cases of OSCC. Multivariate analysis revealed that the risk of malignant progression in the patients with Bmi1-positive expression was significantly higher than those with Bmi1 negativity (odds ratio, 20.75; 95% confidence interval, 2.21-194.57; P = .008). Collectively, Bmi1 expression was significantly associated with malignant transformation in a large series of patients with OLP who received a longitudinal observation. Our findings suggested that Bmi1 may serve as a useful marker for the identification of a high risk of malignant progression of OLP. Crown Copyright © 2013 Published by Elsevier Inc. All rights reserved.
1. Introduction Oral lichen planus (OLP) is a relatively common inflammatory mucocutaneous disease of unknown etiology. It has a protracted clinical course despite various available treatments [1,2]. Patients with OLP carry a significantly increased risk of developing oral squamous cell carcinomas (OSCCs) [3]. Development of OSCC could arise from all clinical types of OLP, with a rate of malignant progression up to 6.4% [4]. Oral lichen planus has been defined as one of potentially malignant disorders by the World Health Organization [5], but clinical and histologic features of OLP lesions have limited prognostic value for malignant progression. Previous cross-sectional studies reported that desmocollin-1, matrix metallopeptidase, Smad, p53, and p16 may be relevant to malignant progression of OLP [6–10]. Obviously, the longitudinal observation
⁎ Corresponding authors. Tel.: +86 21 23271699; fax: +86 21 63087076. E-mail addresses: [email protected] (L. Ma), [email protected] (W. Liu). 1 L. Ma and H. Wang contributed equally to this work.
study on molecular event of the progression is of more scientific and clinical significance [11]. Bmi1 is a polycomb group transcription repressor that mediates gene silencing by regulating chromatin structure. It is considered to be a stem cell–related gene that has been demonstrated to play a role in the self-renewal of cancer stem cells [12]. Bmi1 has been demonstrated to be up-regulated in a subpopulation of cancer stemlike cells isolated from head and neck cancer cells [13–15]. Furthermore, it has been demonstrated to be a prognostic marker in several human cancers including gastric cancer and head and neck cancer [16–18]. Bmi1 was also found to be overexpressed in human gastrointestinal and oral dysplasias, indicating that its abnormal expression occurs early in gastrointestinal and oral carcinogenesis [19,20]. In the current study, we hypothesized that expression of Bmi1 protein would be dysregulated in patients with OLP. In this retrospective follow-up study, we examined protein expression of Bmi1 in samples of nonprogressing OLP, progressing OLP to postmalignant OSCC, and determined the correlation between Bmi1 expression in OLP and the risk of OSCC.
1092-9134/$ – see front matter. Crown Copyright © 2013 Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.anndiagpath.2013.03.002
Please cite this article as: Ma L, et al, Bmi1 expression in oral lichen planus and the risk of progression to oral squamous cell carcinoma, Ann Diagn Pathol (2013), http://dx.doi.org/10.1016/j.anndiagpath.2013.03.002
2
L. Ma et al. / Annals of Diagnostic Pathology xxx (2013) xxx–xxx
Table 1 Characteristics of patients with OLP Characteristic, n (%)
Nonprogressing OLP
Progressing OLP
Patients Follow-up (mo) Mean Range Age (y) Mean (SD) Range Sex Female Male Smoking history Never Past and present Unknown Alcohol intake Never Past and present Unknown Bmi1 expression Negative Positive
87 Time 1 53 16-175
9 Time 2 55 10-132
46.4 (11.7) 26-74
54.9 (8.9) 41-70
66 (75.9) 21 (24.1)
8 (88.9) 1 (11.1)
65 (81.2) 15 (18.8) 7 (−)
6 (75.0) 2 (25.0) 1 (−)
61 (77.2) 18 (22.8) 8 (−)
6 (75.0) 2 (25.0) 1 (−)
55 (63.2) 32 (36.8)
1 (11.1) 8 (88.9)
P .808
.038
.341
.648
1.00
1990 to 2010. Information regarding age, sex, smoking, and alcohol use at the time of the initial diagnosis of OLP was documented. All the study patients underwent biopsy. The biopsy was fixed in formalin, embedded in paraffin, and processed for routine histopathologic examination. The modified World Health Organization criteria for OLP [21] were used when examining the histopathology of the sections. In our clinic, periodic follow-up examinations at intervals of every 6 months or less were recommended for patients with OLP. Patient with nonprogressing OLP (n = 87) was defined as the one who did not progress to OSCC in our follow-up period, and patient with progressing OLP (n = 9) was defined as that subsequently progressed to OSCC confirmed by histopathology at the same oral site of OLP lesion in the follow-up. Ten samples of normal oral mucosa identified in biopsies taken for unrelated benign lesions were used as controls. Six samples of paraffin blocks of postmalignant OSCC lesions from previously diagnosed OLP were also studied. This study was approved by the local institutional review board.
.004
Time 1: From the initial diagnosis of OLP to the last consultation. Time 2: From the initial diagnosis of OLP to progress to carcinoma.
2. Materials and methods 2.1. Patients and tissue specimens A series of 96 patients with the clinical and pathologic diagnosis of OLP in this study were retrieved among the archived files at the Department of Oral Mucosal Diseases, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, from
2.2. Tissue processing and immunohistochemistry Four-micrometer-thick tissue sections from formalin-fixed, paraffin-embedded tissue blocks of the tissue samples were mounted on positively charged glass slides. Immunohistochemical staining was performed using Leica Automatic Stainer (Leica Microsystems Ltd, Wetzlar, Germany) following manufacturer's protocol. The anti-Bmi1 monoclonal antibody (1:150 dilution, ab14389; Abcam, Cambridge, UK) was used to detect Bmi1 expression. In negative controls, the primary antibody was replaced by nonimmune immunoglobulin G of the same isotype to ensure specificity. Cell nuclear immunoreactivity in the epithelium was considered to indicate positive expression of Bmi1. Sample evaluation of immunostaining was performed by oral pathologists (JL and LZW) blinded for the clinical data. According to
Figure. Representative expression pattern of Bmi1. Bmi1-negative expression in normal oral mucosa (A) and nonprogressing OLP (B). Magnification, ×200. Bmi1-positive expression in progressing OLP (C) and post-malignant OSCC from OLP (D). Magnification, ×400.
Please cite this article as: Ma L, et al, Bmi1 expression in oral lichen planus and the risk of progression to oral squamous cell carcinoma, Ann Diagn Pathol (2013), http://dx.doi.org/10.1016/j.anndiagpath.2013.03.002
L. Ma et al. / Annals of Diagnostic Pathology xxx (2013) xxx–xxx
3
4. Discussion
Table 2 Logistic regression analysis of the risk of oral cancer
In this retrospective follow-up study, the 96 patients with OLP were grouped as nonprogressing OLP (n = 87) and progressing OLP (n = 9). The baseline characteristics of these patients are presented in Table 1. A significant difference with regard to age (P = .038, Student t test) was found, whereas differences in follow-up period, sex, smoking history, and alcohol intake were not observed between the 2 groups. The representative immunoexpression of Bmi1 in normal oral mucosa, nonprogressing OLP, progressing OLP, and OSCC form previously diagnosed OLP were shown in the Figure. We observed that Bmi1-positive expression was absent in 10 samples of normal oral mucosa, but it expressed in the 6 cases (100%) of OSCC from OLP. Bmi1 expression was found in 32 (36.8%) of 87 cases of nonprogressing OLP and in 8 (88.9%) of 9 cases of progressing OLP (P = .004, Fisher exact test). Bmi1 expression was significantly increased from normal oral mucosa (0%), nonprogressing OLP (36.8%), progressing OLP (88.9%) to OSCC (100%) (P b .001, Fisher exact test).
Increasing evidence indicates that the initiation, progression, recurrence, and metastasis of head and neck squamous cell carcinoma are related to the behavior of a small subpopulation of cancer stem cells [22–24]. Clinical and therapeutic implications of cancer stem cells have attracted growing attention, including early detection and prognostication of cancer. In addition, cancer stem cells can be identified and isolated by expression of distinctive markers to enrich for stem cells [22–24]. Bmi1 is one of the well-conducted studied cancer stem cells factors, which has been implicated in several solid tumor types including head and neck squamous cell carcinoma [12]. Bmi1 has been studied as an oncoprotein associated with poor prognosis in several human cancers [16–18]. Recent studies have reported that Bmi1, as one of the stem cell markers, was essential for maintaining the self-renewal properties of and tumor progression in head and neck cancer [25–27]. Elevated Bmi-1 expression was demonstrated to be associated with dysplastic cell transformation during oral carcinogenesis and to be required for cancer cell replication and survival [20]. In the previous study, we reported Bmi1 abnormal expression in samples of oral leukoplakia [28] and suggested that it is a predictor for transformation and tumorigenicity of OSCC. In the current follow-up study, we attempted to determine Bmi1 expression pattern in samples of a series of patients with OLP and analyze the correlation between it expression and malignant potential of OLP. One unique feature of the current study is the 2-stage experimental design, which incorporated testing and validation steps. Oral carcinogenesis is a multistep process of cumulative genetic and epigenetic aberrations. First, we observed that Bmi1 expression was significantly increased from normal oral mucosa, nonprogressing OLP, progressing OLP to OSCC. Bmi1 expression level was consistent with malignant potential of oral mucosa, which may reflect a stepwise transformation of OLP into OSCC. Second, we focused on differential expression of Bmi1 in nonprogressing OLP and progressing OLP and association with the risk of malignant progression. The results showed that Bmi1 expression is of significant difference between the 2 groups, and the risk of malignant progression in the patients with Bmi1positive expression was significantly higher than those with Bmi1negative expression. Collectively, these data not only support that the Bmi1 might play a critical role in transformation and tumorigenicity of OSCC but also suggest that it was a potential biomarker for early detection of OLP malignant progression, although the biological function of Bmi1 as a putative cancer stem cell marker in oral potentially malignant lesions remains unclear. In summary, Bmi1 expression was significantly associated with malignant progression in a series of patients with OLP who received a long-term follow-up. Our findings suggested that Bmi1 may serve as a useful marker for the identification of a high risk of oral potentially malignant lesions progressing to OSCC.
3.2. Bmi1 expression in OLP and risk of OSCC
Acknowledgments
To evaluate the risk of OSCC in patients with OLP, Bmi1 expression and clinical characteristics of the patients in the current study were analyzed by logistic regression (Table 2). In the univariate analysis, age (OR, 1.07; 95% CI, 1.00-1.13; P = .047) and Bmi1 expression (OR, 13.75; 95% CI, 1.64-115.02; P = .016) were significantly associated with increased risk of malignant transformation of OLP. To further assess the influence of patient' age, we then did a multivariate analysis. After adjusting for age, Bmi1 expression retained statistical significance. The risk of malignant progression in the patients with Bmi1-positive expression was significantly higher than those with Bmi1-negative expression (adjusted OR, 20.75; 95% CI, 2.21-194.57; P = .008).
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. This study was supported by Shanghai Health Bureau (ZYSNXD-CC-ZDYJ004) and Science and Technology Commission of Shanghai (11DZ1972600).
Characteristic Univariate analysis Age Sex Smoking Alcohol intake Bmi1 expression Multivariate analysis Age Bmi1 expression
OR (95% CI)
P
1.07 (1.00-1.13) 0.39 (0.05-3.33) 1.44 (0.27-7.88) 1.13 (0.21-6.09) 13.75 (1.64-115.02)
.047 .391 .671 .887 .016
1.09 (1.01-1.17) 20.75 (2.21-194.57)
.021 .008
criteria of staining intensity described previously by Kang et al [20], we classified negative or barely detectable epithelium as Bmi1negative expression and weak, moderate to strong staining of epithelium as Bmi1-positive expression.
2.3. Statistics Statistical analysis was carried out with the χ 2 test or Fisher exact test among qualitative variables and the Student t test among quantitative variables. Logistic regression was applied to evaluate relative risk for OLP malignant transformation. Odds ratio (OR) with 95% confidence interval (95% CI) and P values were reported. All tests were 2 sided, and P b .05 was accepted for statistical significance.
3. Results 3.1. Patient characteristics and Bmi1 expression
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Please cite this article as: Ma L, et al, Bmi1 expression in oral lichen planus and the risk of progression to oral squamous cell carcinoma, Ann Diagn Pathol (2013), http://dx.doi.org/10.1016/j.anndiagpath.2013.03.002
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Please cite this article as: Ma L, et al, Bmi1 expression in oral lichen planus and the risk of progression to oral squamous cell carcinoma, Ann Diagn Pathol (2013), http://dx.doi.org/10.1016/j.anndiagpath.2013.03.002