CD8+ and CD163+ infiltrating cells and PD-L1 immunoexpression in oral leukoplakia and oral carcinoma.

Overexpression of inhibitory checkpoint PD1/PD-L1 plays an important role in carcinogenesis and patients prognosis. 70 cases of oral squamous cell carcinoma (OSCC), 23 cases of oral leukoplakia (OLK), and 19 control cases were immunohistochemically stained with anti-PD-L1, -CD8, and -CD163 antibodies. PD-L1 was expressed on dysplastic and subepithelial infiltrating cells of OLK as well as on cancer and tumor-infiltrating cells of OSCC. In OSCC, PD-L1 immunoexpression was significantly increased in comparison to OLK, and control groups. The correlative study showed significant correlations between the immunoexpression of PD-L1 and the number of CD8+, CD163+ cells in both OLK and OSCC groups. We found also significant negative correlation between the number of PD-L1+ infiltrating cells and the number of CD8+ cells in OSCC, and positive correlation between the number of PD-L1+ infiltrating cells and CD163+ cells in OLK and OSCC groups. In conclusion, our study indicate that CD163+ and CD8+ infiltrating cells influence the early and subsequent stages of oral carcinogenesis. We demonstrated also that studied tumors may evade the host immune system by PD-L1 immunoexpression not only on epithelial cells but on infiltrating cells as well.

T cells are involved in the induction of macrophage phenotypes in oral leukoplakia and squamous cell carcinoma-a preliminary report.

BACKGROUND: The prognosis of human malignancies has been shown to depend on immunological parameters, such as macrophage polarization (M1 and M2). In this study, we identify the phenotype of macrophages, and investigate an involvement of infiltrated T cells that participate in the polarization of macrophages, in oral leukoplakia (OLK), and oral squamous cell carcinoma (OSCC). METHODS: Immunohistochemical method was used to examine the number of CD68+ , CD163+ (M2), iNOS+ (M1) macrophages, and CD4+ , CD8+ , CCR4+ (Th2), CCR5+ (Th1) cells in 102 cases of OSCC: without metastases-OSCC M(-) (n = 54), and with metastases-OSCC M(+) (n = 48), 23 cases of OLK, and 18 control cases. RESULTS: The mean number of CD68+ , CD163+ , iNOS+ , CD4+ , CCR4+ , CCR5+ cells was significantly increased in OSCC M(+) group compared with OLK, OSCC M(-) and control group. We found positive correlations between the number of CD4+ T cells and CD163+ and iNOS+ macrophages as well as CCR4+ and CCR5+ cells in both OSCC groups. The mean number of CD8+ cells was significantly increased in OSCC M(-) and OLK compared with OSCC M(+) and control group. In OSCC M(+) and OSCC M(-) groups, a negative correlation between the number of CD8+ cells and CD163+ and iNOS+ macrophages was found. CONCLUSIONS: The number and co-localization of lymphocytes and macrophages in OLK and OSCC may indicate that infiltrating cells influence the early and subsequent stage of oral carcinogenesis.

A proteomic approach to compare saliva from individuals with and without oral leukoplakia.

BACKGROUND: Oral leukoplakia is the most common potentially malignant disorder in the oral cavity and can precede carcinoma. This study aimed to identify possible oral leukoplakia salivary biomarkers. METHODS: Unstimulated saliva was collected from participants and protein concentration was determined. Proteins were then precipitated with cold acetone and separated using 2DE over a pH range of 3-10. Spot demarcation and matching were performed and protein identification was done through MS analysis. Oral leukoplakia tissues were submitted to immunohistochemistry analysis for keratin 10 (CK10). A complementary analysis of oral leukoplakias that were not included previously was performed in addition. RESULTS: 226±10 spots were identified in oral leukoplakia 2DE gels, and 262±12 spots were identified in volunteers. Twenty-two spots were highly abundant in oral leukoplakias or not detected in the control group, such as apolipoprotein A1, alpha amylase, cystatins, keratin 10, and lysozyme precursor. All were identified. All oral leukoplakia cases were immunopositive for CK10, mainly in the superficial epithelial layers. CONCLUSIONS: The 2DE salivary protein profiles of individuals with and without oral leukoplakia were observably different. CK10 appears to be an interesting protein and should be further studied in oral carcinogenesis. SIGNIFICANCE: MS-based proteomics enables large-scale analysis of proteins. Proteomics can provide detailed descriptions of proteomes of cells and tissues, including body fluids, and appears as a powerful tool to study human disorders. Saliva is readily accessible through non invasive collection and can mirror diverse disease states. Saliva from both diseased and healthy subjects can be analyzed through 2DE and differences between groups could be found. Routine immunohistochemistry analysis confirmed one of these findings, with CK10 being positive tissues from individuals with oral leukoplakia. Therefore, the present study allows insights into development of an important potential oral cancer precursor, named oral leukoplakia. However, the results can be extrapolated and tested in other precancer states, such as proliferative verrucous leukoplakia, patients at risk of oral cancer due to lifestyle behavior and/or cancer history in the family or even those who are under surveillance after a treated primary oral cancer.

The influence of sleep restriction on expression of apoptosis regulatory proteins p53, Bcl-2 and Bax following rat tongue carcinogenesis induced by 4-nitroquinoline 1-oxide.

PURPOSE: The aim of this study was to evaluate whether sleep restriction (SR) could affect the mechanisms and pathways' essentials for cancer cells in tongue cancer induced by 4-nitroquinoline 1-oxide in Wistar rats. METHODS: The animals were distributed into 4 groups of 5 animals each treated with 50 ppm 4 NQO solution through their drinking water for 4 and 12 weeks. The animals were submitted to sleep restriction for 21 days using the modified multiple platform method, which consisted of placing 5 rats in a cage (41 × 34 × 16 cm) containing 10 circular platforms (3.5 cm in diameter) with water 1 cm below the upper surface. The investigations were conducted using immunohistochemistry of p53, Bax and Bcl-2 proteins related to apoptosis and its pathways. RESULTS: Although no histopathologic abnormalities were induced in the epithelium after 4 weeks of carcinogen exposure in all groups, in 12 weeks were observed pre-neoplastic lesions. Data analysis revealed statistically significant differences (P < 0.05) in 4 weeks group for p53, and for bcl-2. Following 12 weeks of 4NQO administration, we found significant differences between SR and control groups in p53, bax, and bcl-2 immunoexpression. CONCLUSION: Our results reveal that sleep restriction exerted alterations in proteins associated with proliferation and apoptosis in carcinogenesis.

E-cadherin as a potential biomarker of malignant transformation in oral leukoplakia: a retrospective cohort study.

BACKGROUND: Numerous attempts have been made to establish and develop tumor markers that could determine the susceptibility of normal tissues to transform into cancerous ones. To determine whether altered expression patterns of E-cadherin could be an early event in the progression of potentially malignant disorders to oral squamous cell carcinoma, this study aimed to assess the relationship between the immunoexpression of E-cadherin and the different degrees of epithelial dysplasia in oral leukoplakia. METHODS: Surgically excised specimens from patients with oral leukoplakia (n=31), oral cavity squamous cell carcinoma with cervical lymph node metastasis (n=12) and normal oral mucosa (n=9) were immunostained for E-cadherin. Oral leukoplakia samples were distributed into low and high risk group according to a binary system for grading oral epithelial dysplasia. Comparative analyses between E-cadherin expression and microscopic features (WHO histological grading and epithelial dysplasia) were performed by Pearson Chi-square test (P<0.05). RESULTS: Differences in E-cadherin expression were observed between normal oral mucosa and low risk oral leukoplakia (P=0.006), low and high risk oral leukoplakia (P=0.019), and high risk oral leukoplakia and oral cavity squamous cell carcinoma with cervical lymph node metastasis (P=0.0001). In addition, as epithelia undergo dysplastic changes, the risk of malignant transformation increases, and there is a reduction or loss of E-cadherin expression by keratinocytes. Reduced E-cadherin expression was an early phenomenon and it was observed in moderate-severe dysplasia, showing that the loss of epithelial cohesion may be an indicator of progression to oral cavity squamous cell carcinoma. CONCLUSIONS: E-cadherin could be used as a novel biomarker to identify lesions with potential risk for malignant transformation, which may provide opportunities for prophylactic interventions in high risk patient groups.

MCM3 could be a better marker than Ki-67 for evaluation of dysplastic oral lesions: an immunohistochemical study.

BACKGROUND: The aim of this study was to identify the expression of MCM3, Ki-67 and p27 in normal mucosa, leucoplakia and oral squamous cell carcinoma (OSCC) and determine whether altered expression could serve as a prognostic marker of a malignant progression of dysplastic lesions. METHODS: The samples were collected from 37 patients with oral leucoplakia (13 with mild dysplasia - MLD, 12 with moderate dysplasia - MD and 12 with severe dysplasia - SD). Eleven samples of mouth floor mucocele (M) and 50 floor mouth and tongue samples OSCC of untreated patients were included in this study. Immunohistochemical expression of MCM3, Ki-67 and p27 of all the groups was analysed. Kruskal-Wallis and Dunn's test were used to determine differences among groups, and a Pearson's correlation test was used to evaluate the correlation between the proteins. RESULTS: Ki-67 expression was higher in OSCC than M (P < 0.001) and MLD (P < 0.01) groups, and there was a lower expression in M compared with MD and SD (P < 0.05). Regarding p27, its expression was lower in OSCC compared with M, MD and SD. MCM3 expression was lower in M compared with SD and OSCC (P < 0.001), and MLD showed a lower expression when compared SD (P < 0.01) and OSCC (P < 0.001). Moreover, a better correlation was observed between the proteins MCM3 and p27 than between Ki-67 and p27 proteins when all lesions were examined together. CONCLUSIONS: This study showed that MCM3 could be a better marker than Ki-67 for evaluation of dysplastic oral lesions.

Immunohistochemical detection of p53 and p63 in oral squamous cell carcinoma, oral leukoplakia, and oral submucous fibrosis.

AIM: Oral squamous cell carcinoma (OSCC) may be preceded by potentially malignant disorders such as leukoplakia and oral submucous fibrosis (OSF). p63 in concert with p53 regulates cell proliferation and differentiation and may have a role in potentially malignant and malignant lesions of the oral cavity. The aim of the study was to evaluate and compare the expression of p53 and p63 proteins in OSCC, leukoplakia, and OSF by immunohistochemistry (IHC). METHODS: Tissue sections of OSCC (n = 20), leukoplakia (n = 20), OSF (n = 20) and normal oral mucosa (n = 10) were stained with p53 and p63 antibodies by IHC. Mean labeling index (LI) among the study groups were compared using the Kruskal-Wallis test. RESULTS: The mean LI of p53 for OSCC, leukoplakia, OSF, and normal mucosa were 56.9 ± 21.3, 37.6 ± 12.6, 34.6 ± 8.7 and 15.1 ± 9, while mean LI of p63 were 56.8 ± 19.6, 42.3 ± 10.5, 32.8 ± 12.1, and 26.4 ± 9.4. The mean LI of p53 and p63 were significantly higher in OSCC, leukoplakia and OSF compared to normal mucosa (P < 0.01; P < 0.01). CONCLUSION: The significant increase in expression of p53 and p63 proteins in OSCC, leukoplakia, and OSF suggests their role as surrogate markers of malignant transformation.

Relationship between microRNA expression levels and histopathological features of dysplasia in oral leukoplakia.

BACKGROUND: Increased expression of microRNAs (miRNAs), miR-21, miR-345, and miR-181b has been demonstrated in oral leukoplakia (OL) that progresses to oral squamous cell carcinoma (OSCC), suggesting a miRNA signature with potential prognostic value. On the basis of these findings, this pilot study aimed to investigate the cytological and histopathological features that are used to grade oral dysplasia and determine associations with the expression of these 3 potentially cancer-related miRNAs. We also compared the expression levels of these miRNAs in OL with normal oral mucosa and OSCC. METHODS: We evaluated miRNA expression by qPCR in 22 samples of OL demonstrating different grades of dysplasia, as well as 17 cases of OSCC, and 6 samples of normal oral mucosa. We associated the miRNAs expression profiles with cytological and histopathological features of OL. RESULTS: OSCC cases showed increased expression of all 3 miRNAs when compared with OL and normal oral mucosa. Increased expression of miR-21 was also observed in OL when compared with normal oral mucosa. We found a higher expression of miR-21 and miR-181b in OL that presented with an increased number of mitotic figures, increased nuclear/cytoplasmic ratio, or hyperchromasia. Increased expression of miR-21 was also detected in OL with abnormally superficial mitosis. Higher expression of miR-345 was observed in OL with an increased number and size of nucleoli or increased nuclear/cytoplasmic ratio. CONCLUSIONS: In conclusion, the present study shows that some cytological and histopathological parameters used to grade dysplasia are associated with altered miRNA expression.

STAG2 expression in oral cancer and potentially malignant lesions.

Oral cancer is a world health problem, and one of the highest incidence rates of oral cancer worldwide occurs in Brazil. STAG2 is part of the cohesin complex which is responsible for sister chromatid cohesion. STAG2 loss of expression was reported in a range of tumors, and STAG2 loss was found to cause chromosomal instability and aneuploidy in cancer cells. On the basis of these findings, we investigated STAG2 expression in oral cancer and potentially malignant lesions. We investigated STAG2 immunoexpression in oral cancer, lip cancer, oral leukoplakia, and actinic cheilitis, including complete clinical information. Normal oral mucosa samples were included as normal controls. STAG2 protein was highly expressed in all samples. We further tested STAG2 expression in gastric adenocarcinomas and glioblastomas, as these tumor types were previously shown to lose STAG2 expression. We found homogenous expression of STAG2 by these tumor cells. Our results suggest that STAG2 loss of expression is not a common event in oral carcinogenesis.

Overexpression of Smad proteins, especially Smad7, in oral epithelial dysplasias.

OBJECTIVE: Transforming growth factor ß, via membrane-bound receptors and downstream Smad2-4, 7, can modulate tumorigenesis. Smad2 and Smad3 heterodimerize with Smad4, and the complex migrates to the nucleus to regulate the expression of target genes. Smad7 is a key negative regulator of this signaling pathway. This study aimed to examine Smad2-4, 7 expression and phosphorylated Smad2-3 (p-Smad2-3) in oral epithelial dysplasia and compared it with normal oral mucosa, hyperkeratosis/epithelial hyperplasia and squamous cell carcinoma (SCC). MATERIALS AND METHODS: Immunohistochemical staining of Smad2-4, 7 and p-Smad2-3, was performed for 75 samples of human oral mucosa, including hyperkeratosis/epithelial hyperplasia (n = 20), mild epithelial dysplasia (n = 11), moderate to severe epithelial dysplasia (n = 11), and SCC (n = 43). Normal buccal mucosa samples (n = 9) were also included. RESULTS: A significant increase in Smad7 expression was observed in the ascending order of samples of normal oral mucosa, hyperkeratosis/epithelial hyperplasia/mild oral epithelial dysplasia, moderate to severe oral epithelial dysplasia, and well-differentiated oral SCC/moderately to poorly differentiated oral SCC. Additionally, significant increases in Smad7 expression were noted as compared with expression of Smad2-4 and p-Smad2-3 in lesions of hyperkeratosis/epithelial hyperplasia, mild oral epithelial dysplasia, moderate to severe oral epithelial dysplasia, well-differentiated oral SCC, and moderately to poorly differentiated oral SCC. CONCLUSIONS: Our results indicate that Smad proteins, particularly Smad7, in oral epithelial dysplasia and SCC could contribute to the attenuation of Smads anti-proliferative signaling in cancer development. CLINICAL RELEVANCE: Smad7 could be a marker for risk of malignant transformation of oral epithelial dysplasia.

Expression of the cell cycle regulation proteins p53 and p21WAF1 in different types of non-dysplastic leukoplakias.

OBJECTIVES: The aim of this study was to analyze the immunolabeling of two cell cycle protein regulators, p53 and p21WAF1, in non-dysplastic leukoplakias with different epithelial alterations: acanthosis, hyperkeratosis and acanthosis combined with hyperkeratosis, and compare them with dysplastic leukoplakias. MATERIAL AND METHODS: This was a prospective cohort study involving 36 patients with oral homogeneous leukoplakias. excisional biopsies were performed and the patients remain under clinical follow-up. The leukoplakias were divided into four groups: 6 acanthosis, 9 hyperkeratosis, 10 acanthosis combined with hyperkeratosis, and 11 epithelial dysplasias. Paraffin-embebeded sections were immunostained for p53 and p21WAF1. Five hundred cells from the basal layer and 500 from the parabasal layer were counted to determine the percentage of positive cells. A qualitative analysis was also carried out to determine the presence or absence of immunohistochemical staining in the intermediate and superficial layers. Groups were compared with ANOVA (p<0.05). Pearson's correlation coefficient was used to test for associations between the two markers, p53 and p21WAF1. RESULTS: No leukoplakia recurred and no malignant transformation was observed whitin a follow-up period of 3-6 years. The mean percentage of p53 staining in the basal and parabasal layers was similar in all groups. p21WAF1 staining differed between layers was as follows: in the basal, only 3 to 4% of cells were stained, while in the parabasal, between 16 and 28% of the epithelial cells were stained in the four different studied groups with no statistically significant difference (p>0.05). CONCLUSIONS: Our findings failed to differentiate the non-dysplastic lesions by means of p53 and p21WAF1 immunostaining, notwithstanding similar profiles between non-dysplastic and dysplastic leukoplakias were observed.

Coexpression of p53 and Ki 67 and lack of c-erbB2 expression in oral leukoplakias in India

Oral cancer is commonly preceded by premalignant lesions and conditions. The clinician's ability to identify lesions at an increased risk of cancer development is critical for its control. The purpose of this study was to compare the expression of tumor suppressor gene p53, proliferation marker Ki-67, and oncogene c-erbB2 and to evaluate the relevance of their co-expression in the diagnosis of, and prognosis for, oral leukoplakia. In the present study, the expression of biomarkers was studied immunohistochemically in 55 cases of leukoplakia (26 without dysplasia, 29 with dysplasia) and 10 cases of normal epithelia. The Labeling Indices (LI) of p53 and Ki-67 were found to increase significantly with an increase in the grade of dysplasia. A significant correlation was also found between the LI of p53 and that of Ki-67. It was also observed that c-erbB2 expression was only cytoplasmic, indicating incomplete receptor degradation. Hence, it can be concluded from the present study that the increased expression of p53 and Ki-67 with an increase in the grade of dysplasia suggests that their co-expression may be used for the identification of high-risk lesions. Also, c-erbB2 has no pathogenetic role in early carcinogenesis in the studied population, although incomplete receptor degradation, as evidenced by cytoplasmic staining, may indicate an early change.

Expression of the cell cycle regulation proteins p53 and p21WAF1 in different types of non-dysplastic leukoplakias

OBJECTIVES: The aim of this study was to analyze the immunolabeling of two cell cycle protein regulators, p53 and p21WAF1, in non-dysplastic leukoplakias with different epithelial alterations: acanthosis, hyperkeratosis and acanthosis combined with hyperkeratosis, and compare them with dysplastic leukoplakias. MATERIAL AND METHODS: This was a prospective cohort study involving 36 patients with oral homogeneous leukoplakias. excisional biopsies were performed and the patients remain under clinical follow-up. The leukoplakias were divided into four groups: 6 acanthosis, 9 hyperkeratosis, 10 acanthosis combined with hyperkeratosis, and 11 epithelial dysplasias. Paraffin-embebeded sections were immunostained for p53 and p21WAF1. Five hundred cells from the basal layer and 500 from the parabasal layer were counted to determine the percentage of positive cells. A qualitative analysis was also carried out to determine the presence or absence of immunohistochemical staining in the intermediate and superficial layers. Groups were compared with ANOVA (p<0.05). Pearson's correlation coefficient was used to test for associations between the two markers, p53 and p21WAF1. RESULTS: No leukoplakia recurred and no malignant transformation was observed whitin a follow-up period of 3-6 years. The mean percentage of p53 staining in the basal and parabasal layers was similar in all groups. p21WAF1 staining differed between layers was as follows: in the basal, only 3 to 4% of cells were stained, while in the parabasal, between 16 and 28% of the epithelial cells were stained in the four different studied groups with no statistically significant difference (p>0.05). CONCLUSIONS: Our findings failed to differentiate the non-dysplastic lesions by means of p53 and p21WAF1 immunostaining, notwithstanding similar profiles between non-dysplastic and dysplastic leukoplakias were observed.

Coexpression of p53 and Ki 67 and lack of c-erbB2 expression in oral leukoplakias in India.

Oral cancer is commonly preceded by premalignant lesions and conditions. The clinician's ability to identify lesions at an increased risk of cancer development is critical for its control. The purpose of this study was to compare the expression of tumor suppressor gene p53, proliferation marker Ki-67, and oncogene c-erbB2 and to evaluate the relevance of their co-expression in the diagnosis of, and prognosis for, oral leukoplakia. In the present study, the expression of biomarkers was studied immunohistochemically in 55 cases of leukoplakia (26 without dysplasia, 29 with dysplasia) and 10 cases of normal epithelia. The Labeling Indices (LI) of p53 and Ki-67 were found to increase significantly with an increase in the grade of dysplasia. A significant correlation was also found between the LI of p53 and that of Ki-67. It was also observed that c-erbB2 expression was only cytoplasmic, indicating incomplete receptor degradation. Hence, it can be concluded from the present study that the increased expression of p53 and Ki-67 with an increase in the grade of dysplasia suggests that their co-expression may be used for the identification of high-risk lesions. Also, c-erbB2 has no pathogenetic role in early carcinogenesis in the studied population, although incomplete receptor degradation, as evidenced by cytoplasmic staining, may indicate an early change.

IMP3 is a novel biomarker to predict metastasis and prognosis of tongue squamous cell carcinoma.

Insulinlike growth factor II mRNA-binding protein 3 (IMP3) is a newly identified mRNA-binding protein that is involved in embryogenesis and carcinogenesis of some malignant tumors. The aim of this study was to investigate the clinicopathologic significance of this protein in tongue squamous cell carcinoma (SCC). The expression of IMP3 in 65 samples of tongue SCC and 27 cases of oral leukoplakia (OL) was evaluated by immunohistochemistry. These expression levels were correlated with clinical and pathologic features as well as death from tongue SCC. Weak immunohistochemical stain for IMP3 was identified in all 19 cases of OL with mild dysplasia, and no immunohistochemical reactivity was found in 8 cases of OL without dysplasia. Positive immunohistochemical stain for IMP3 was identified in 50 cases (77%) of SCC; among them, weak staining was identified in 33 cases (51%) and intermediate staining in 17 cases (26%). To compare the expression of IMP3 in tongue SCC and OL, stronger immunohistochemical reactivity was found in tongue SCC (P < 0.05). Stronger expression of IMP3 was found to be associated with lymphoid metastasis (P < 0.05) and patient poor outcome (median survival time of 40 months in the negative and weak expression group vs 10 months in the intermediate expression group; P < 0.05). This study suggests that the increase in IMP3 expression in tongue leukopathia and SCCs may play a role in the carcinogenesis and tumor metastasis of tongue SCCs. Insulinlike growth factor II mRNA-binding protein 3 could be a novel prognostic indicator for patients with tongue SCCs.

Diagnostic efficiency of toluidine blue with Lugol's iodine in oral premalignant and malignant lesions.

BACKGROUND AND OBJECTIVES: In vivo stains are prompt resources, which have emerged, in the recent years, to aid as clinical diagnostic tools in detecting early premalignant and malignant lesions. The aim of the study was to determine the diagnostic efficiency of toluidine blue with Lugol's iodine in oral premalignancies and malignancies and to evaluate the reliability of in vivo staining with toluidine blue and Lugol's iodine in the lesions at risk of malignancy. MATERIALS AND METHODS: The study group comprised 30 subjects with clinically suspicious premalignant lesions and 30 subjects with clinically suspicious malignant lesions. All the lesions were stained consecutively with toluidine blue and Lugol's iodine and the dye retention were recorded with photographs. Depending on the retention of the dyes, the biopsy site was determined. The biopsy specimens were sent for histological confirmation and results were statistically analyzed. RESULTS: The overall diagnostic accuracy of Lugol's iodine when used consecutively with toluidine blue stain in distinguishing premalignant lesions and malignant lesions was 90%. As the degree of differentiation of malignant lesions progressed toward more severity, they failed to show the retention of Lugol's iodine and the result was highly significant statistically, with a P value < 0.001. INTERPRETATION AND CONCLUSION: Lugol's iodine when used with toluidine blue helped in delineating the inflammatory lesions and was the mean source in determining clinically the degrees of differentiation of malignant lesions as the poorly differentiated malignant lesions without glycogen content failed to show Lugol's iodine retention. Toluidine blue with Lugol's iodine can be used as a pretherapeutic assessment of the biologic aggressiveness of the disease.

Detection of survivin mRNA in healthy oral mucosa, oral leucoplakia and oral cancer.

BACKGROUND: Survivin is involved in modulation of cell death and cell division processes. Survivin expression in normal adult tissues has not been fully understood, although it is markedly lower than in cancer, where it is over-expressed. OBJECTIVE: To investigate survivin expression in normal, potentially malignant and cancerous oral mucosa. METHODS: We measured survivin mRNA levels by real-time RT-PCR in specimens of oral mucosa (15 from normal mucosa, 17 from potentially malignant lesions, 17 from neoplasms). Scores were compared using Kruskal-Wallis test and post hoc according to Conover. Chi-squared test was used for dichotomous data. RESULTS: The median relative levels of survivin mRNA resulted six for normal mucosa, eight for potentially malignant lesions, 13 for cancers: differences among these three groups were statistically significant, as between cancer and potentially malignant lesions. Expression in normal mucosa and potentially lesions group showed no significant difference. Low, but not marginal expression of survivin in normal mucosa is a new finding, and it could be explained with the higher sensibility of our methods. CONCLUSIONS: Survivin expression in oral potentially malignant lesions might indicate a progressive deregulation of expression paralleling oncogenesis, particularly during the first stages of process, suggesting a putative predictive role for survivin.

Keratin 8 expression in head and neck epithelia.

BACKGROUND: The intermediate filament forming protein keratin 8 (K8) is a tumour-associated antigen, which was shown to be over-expressed in a variety of malignancies. Here, we present a study of K8 expression in squamous epithelia of the head and neck area, including normal mucosa, hyperplastic and dysplastic leukoplakia, carcinomas of different sub-localisations, and lymph node metastases. METHODS: K8 expression was assessed upon immunohistochemistry with specific antibodies in cryosections of primary tumours of the head and neck area. RESULTS: K8 expression was characteristic of transformed tissue and marked early stages of disease, i.e. dysplastic oral leukoplakia, but not normal or hyperplastic epithelium. With the exception of carcinomas of the larynx and the tongue, K8 expression also strictly differentiated carcinomas from normal epithelium of the same origin. Furthermore, K8high was characteristic of cells, which had detached from the sites of primary tumours and had been invading the surrounding tissue at the time point of surgery. CONCLUSION: K8 is an excellent marker for head and neck malignancies, which allows for early detection as well as for visualisation of potentially disseminating tumour cells in vivo.

Argyrophilic nucleolar organizer regions in inflammatory, premalignant, and malignant oral lesions: a quantitative and qualitative assessment.

BACKGROUND AND OBJECTIVE: Argyrophilic nucleolar organizer regions (AgNORs) have found widespread application in the past, especially in tumor histopathology. This study was undertaken to evaluate the significance of various AgNOR parameters and to assess their role in differentiating hyperplastic, premalignant, and malignant lesions. MATERIALS AND METHODS: The study sample consisted of archival biopsy specimens of ten squamous cell carcinomas, ten premalignant lesions, and five inflammatory lesions. Two biopsies from normal mucosa acted as control. AgNORs were assessed both quantitatively and qualitatively. The data were analyzed using Student's independent t-test, one-way analysis of variance (ANOVA), and multiple range test (Tukey-HSD). RESULTS: Quantitatively significant difference existed in the number of AgNORs between the normal mucosa, inflammatory lesions, and carcinomas, but the premalignant lesions failed to differ significantly from the normal mucosa. The number of AgNORs was found to be related to epithelial proliferation. Qualitatively, in terms of size, shape, and pattern of distribution, the normal mucosa and inflammatory lesion were alike, but the premalignant and malignant lesions differed significantly from the normal, with a marked degree of AgNOR pleomorphism being observed in carcinomas. CONCLUSIONS: AgNOR quantity is strictly proportional to the proliferative activity of the cell and does not necessarily indicate malignancy. It is the qualitative characteristics of AgNOR that help to differentiate hyperplastic, premalignant, and malignant lesions.

Predicting recurrence after oral precancer treatment: use of cell cycle analysis.

Prediction of the behaviour of oral precancerous lesions (OPLs) is unreliable in clinical practice. The aim of this study was to analyse the efficacy of cell cyclin markers A and B1, and the proliferative marker Ki67, in predicting clinical outcome for patients with OPLs. A cohort of previously-treated patients with single OPLs were retrieved from the MaxilloFacial Dysplasia database and reviewed. All had dysplastic lesions excised by laser and were followed up for 5 years post-treatment. Outcome was determined as no recurrence or further disease. Excision specimens were re-examined immunohistochemically and labelling indices (LIs) for cyclin A, B1 and Ki67 determined. Forty patients, aged between 31 and 91 years, were recruited. There were no differences in age or sex. OPLs were predominantly leukoplakias on the floor of mouth or ventro-lateral tongue (65%), most of which exhibited moderate or severe dysplasia. Cyclin A LIs ranged from 3.9% to 31.3%, B1 0 to 28.3% and Ki67 3.5% to 54.5%. Using median LIs as 'cut off points' (12% cyclins; 22% Ki67) Kaplan-Meier survival analysis showed a significant risk of further progression of disease in patients with OPL LIs exceeding median values (Cyclin A p=0.02, Cyclin B1 p=0.01, Ki67 p=0.025). By combining analysis of both Cyclin A and B LI, the significance of the difference was increased (p<0.01). Cell cycle analysis is effective in identifying patients at risk of further progression of disease following treatment of OPLs. Multi-centre, longitudinal trials are needed to assess the precise role of cell cycle markers in their management.