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RESEARCH REPORT |
1 Institute of Dental Sciences, Faculty of Medicine, University of Ancona, Ancona, Italy;
2 Department of Biomorphological and Functional Sciences, Pathology Unit, Faculty of Medicine, University of Naples Federico II, Naples, Italy;
3 Institute of Dental Sciences, University of Catania, Catania, Italy;
4 Department of Dental Sciences and Maxillofacial Surgery, Faculty of Medicine, University of Naples Federico II, Naples, Italy;
5 Department of Oral Pathology, Faculty of Dentistry, Hiroshima University, Hiroshima, Japan; and
6 Department of Cancer & Biology and the Cancer Center, Medical School, University of Massachusetts, Worcester, USA;
* corresponding author, Via Carelli 28, 71100 Foggia, Italy, lomuziol{at}tin.it or llomuzio{at}tin.it
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: survivin • precancerous lesion • IAP • oral squamous cell carcinoma • apoptosis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Although up to a third (3-33%) of oral precancerous lesions will eventually evolve into invasive OSCC over a 10-year interval, no reliable histopathological parameters have been identified that predict their potential for subsequent transformation. A prophylactic surgical management is often impractical, especially in patients with multiple and extensive precancerous lesions. Therefore, novel molecular predictors of malignant progression are needed to identify oral precancerous lesions at greater risk of invasive transformation as candidates for surgical intervention.
Considerable interest has focused on the identification of regulators of programmed cell death, or apoptosis, which may influence the cell death/cell viability balance in cancer. In particular, deregulation of apoptosis resulting in aberrantly reduced cell death is thought to participate in cancer by facilitating the insurgence of additional transforming mutations. In addition to pro- and anti-apoptotic bcl-2 molecules, a second gene family of inhibitors of apoptosis (IAP) has recently been identified (Salvesen and Duckett, 2002). Highly evolutionarily conserved from viruses to mammalian cells (Deveraux and Reed, 1999; Salvesen and Duckett, 2002), certain IAP proteins target a downstream step in apoptosis by inhibiting the terminal effectors caspase-3 and -7 and by interfering with processing/activation of the pinnacle caspase, caspase-9 (Deveraux and Reed, 1999; Salvesen and Duckett, 2002).
Survivin is a recently characterized IAP protein (Ambrosini et al., 1997), which is found abundantly expressed in solid and hematological malignancies, but which is undetectable in most normal adult differentiated tissues (Altieri, 2001). Despite the redundancy of cell death pathways, survivin appears to be required for cancer cell viability, and interference with survivin expression/function has been associated with catastrophic defects of mitotic transition and induction of mitochondrial-induced cell death (Altieri, 2001). Survivin may also provide a reliable indicator of disease progression, and retrospective analysis of various solid tumors has linked survivin expression to decreased overall survival, negative predictive indicators of aggressive disease, resistance to therapy, and accelerated rates of recurrence (Altieri, 2001).
In this study, we sought to investigate the potential expression and impact of survivin in oral precancerous lesions, and its potential predictive value for the identification of lesions at high risk of progression to invasive carcinoma.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Immunohistochemistry
Four-µm serial sections from formalin-fixed paraffin-embedded blocks were cut for each case and mounted on poly-L-lysine-coated glass slides. Only sections containing epithelium sufficient for assessment of antibody reactivity with 1000 cells were considered for this study. Immunohistochemical staining was carried out with a rabbit polyclonal antibody supplied by NOVUS (NOVUS Biologicals, Littleton, CO, USA) raised against full-length recombinant survivin characterized in previous studies (Fortugno et al., 2002) after antigen retrieval by pressure cooking and detection by streptavidin-biotin-peroxidase with use of the L.V. Dako LSAB kit (DAKO A/S, Carpinteria, CA, USA) (see Lo Muzio et al., 2001).
An anti-human p27Kip1 mouse monoclonal antibody (diluted 1:100, K25020, Transduction Laboratories, Lexington, KY, USA) and an anti-human p53 mouse monoclonal antibody (diluted 1:100, DO-7, DAKO, Glostrup, Denmark) were used. Moreover, to determine the proliferative cell compartment on consecutive tissue sections and correlate them with survivin expression, we examined the Ki-67 expression by immunohistochemistry using an anti-human Ki-67 mouse monoclonal antibody (diluted 1:50, MIB-1, Immunotech, Marseille, France). The sections were treated with the primary antibodies at 4°C overnight. Negative control slides in the absence of primary antibody were included for each staining. The results of the immunohistochemical staining were evaluated separately by two observers, blind to the histological diagnosis of the single cases and especially to the follow-up data for the respective patients. To evaluate survivin expression in the various samples examined, we applied a scoring method (Lu et al., 1998). A mean percentage of positive tumor cells was determined by the examination of 300 cells in at least five areas at 400x magnification. Cells were assigned to one of the five following categories: (a) 0, < 5%; (b) 1, 5-25%; (c) 2, 26-50%; (d) 3, 51-75%; or (e) 4, > 75%. Cases with weighted scores of less than 1 were weighted as negative. To evaluate p53 expression, we applied a scoring method and assigned a mean percentage of positive tumor cells to one of the two following categories: (a) 0, < 15%; or (b) 1, > 15%. To evaluate Ki-67 expression, we used the following scoring categories: (a) 1, < 5%; (b) 2, > 5-10%; or (c) 3, > 10%. To evaluate p27 expression, we used the following scoring categories: (a) 0, < 5%; (b) 1, > 5-10%; or (c) 2, > 10%.
Statistical Analysis
Data were analyzed with the use of Prism (version 3.0 for Windows) and Stanton A. Glantz (version 3.0 for DOS) software. Statistically significant differences (p < 0.05) between groups were determined by one-way analysis of variance with the Bonferroni Multiple Comparisons Test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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). The magnitudes of survivin expression in progressing and non-progressing lesions were considerably different. Cases of precancerous lesions that did not evolve into OSCC revealed a mean survivin score of 0.63 (Table 2). In contrast, all cases exhibiting malignant transformation showed a mean survivin score of 2.75. The neighboring normal mucosal epithelium showed nuclear expression of survivin only in focal cells of basal and parabasal layers. No statistically significant correlation was found between survivin overexpression and the histological degree of epithelial dysplasia (p > 0.05).
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) and a mean value of 2.93 (Table 2). Survivin expression was observed as a cytoplasmic/nuclear reactivity in cancer cells (Fig. d).
Statistical Analysis
Variations among group means analyzed by one-way analysis of variance were statistically significant (p < 0.001) (Table 3). In contrast, variations between OSCC and dysplasia with subsequent transformation or between dysplasia without subsequent transformation and normal mucosa were not significant (p > 0.05). We compared survivin expression with Ki-67, p27, and p53 expression and found no statistically significant correlation between them (p > 0.05).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Consistent with the data presented here, survivin expression has been shown in other preneoplastic lesions, including polyps of the colon, and nearly all cases of Bowen’s disease (SCC in situ) and hypertrophic actinic keratosis (Chiodino et al., 1999; Grossman et al., 1999). The molecular basis for the selective expression of survivin in malignant and premalignant lesions, as compared with normal tissues, has not been completely elucidated (Altieri, 2001). Although the survivin gene is expressed in mitosis in a cell-cycle-dependent manner (Li et al., 1998), expression of survivin in cancer is seen in virtually all tumor cells, not restricted to the mitotic fraction, suggesting that malignant transformation is associated with global deregulation of survivin gene expression (Altieri, 2001). This is consistent with recent data identifying survivin as one of the genes repressed by wild-type p53 (Hoffman et al., 2002; Mirza et al., 2002; Zhou et al., 2002), suggesting that loss of p53, which is one of the most common genetic abnormalities in cancer), may result in de-repression of survivin gene transcription.
Our cases showed only low expression of p53 in cases of precancerous lesions that did not evolve into OSCC, and in particular in 1/8 cases with mild (12.5%), 1/9 cases with moderate (11,11%), and 3/6 cases with severe dysplasia (50%) (Table 1). In contrast, cases exhibiting malignant transformation showed positivity in 1/4 cases with mild (25%), 3/8 cases with moderate (37.5%), and 1/2 cases with severe dysplasia (50%). Another study on p53 expression in oral precancerous lesions reported similar data, with negativity for p53 in 79% of epithelial dysplasias (Kudo et al., 1999). Other authors reported positivity in 63% of OSCC cases (Schildt et al., 2003) and in the majority of the 31 dysplastic lesions (Choi et al., 2003). An inverse pattern of expression among p27, p53, and Ki-67 in the dysplastic lesions was noted (Choi et al., 2003). p27 inhibits CDK-2 and transforming growth factor-ß (TGF-ß) and has been associated with cell development and differentiation. According to previous studies (Kudo et al., 1999; Choi et al., 2003), p27 expression was evident in the superficial and terminally differentiated cells of histologically nondysplasic squamous epithelium, and in early stages of dysplasia. p27 expression is mainly associated with differentiation in both histologically normal epithelium and tumorigenesis (Fan et al., 1997; Jordan et al., 1998). Some authors reported low p27 expression in poorly differentiated carcinoma, and this correlated significantly with high cellular proliferation as manifested by elevated p53 and Ki-67 markers (Jordan et al., 1998; Mineta et al., 1999; Saito et al., 1999; Choi et al., 2003). Kudo et al.(2000) have suggested that abnormalities in p27 and p53 expression are involved in different phases of squamous tumorigenesis.
In our cases, p27 expression was not correlated with differentiation and/or biological behavior of the lesions. Instead, p53 showed a progressively increased expression from mild to severe oral dysplasias and in infiltrating OSCC with worse clinical behavior. Similarly, Ki67 expression levels were increased in severe dysplasias and OSCC. In summary, deregulation of cell-cycle regulatory proteins cooperatively or independently contributes to SCC development and progression. These results further support a potential utility of these biomarkers in assessing the progression of individual head and neck squamous tumors.
Analysis of the data presented here suggests that this process of survivin re-expression is an early event during stepwise malignant transformation, which may confer selective growth advantage and resistance to environmental or checkpoint-initiated pro-apoptotic stimuli. In our opinion, the overexpression of survivin represents, in all cases, a hallmark of malignant conversion. Naturally, higher scores of survivin expression are likely to be more indicative of this risk. Considering that, in hyperplastic epithelia without associated dysplasia, survivin positivity may reach 5-10% of cells (never exceeding this value), we consider this a possible cut-off value for positivity able to predict malignant conversion.
OSCC is a frequent tumor in humans and carries elevated rates of recurrence (Al-Rajhi et al., 2000) that may involve up to 80% of patients within 2 yrs (Mattijssen et al., 1993). In addition, the five-year survival rates of OSCC are not encouraging (Friedlander et al., 1998). This stresses the need for new molecular markers of disease progression that could reliably identify patients at high risk of developing invasive disease. Consistent with its predictive/prognostic value in other tumors (Altieri, 2001), histological determination of survivin expression in oral premalignant lesions may provide a quick and potentially useful indicator for identifying patients requiring more aggressive therapeutic intervention.
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ACKNOWLEDGMENTS |
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Received January 8, 2003; Last revision August 17, 2003; Accepted August 25, 2003
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REFERENCES |
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