Corrado Rubini

MicroRNA expression profiling of oral carcinoma identifies new markers of tumor progression.

Oral squamous cell carcinoma, the most frequently occurring malignant head and neck tumour, generally exhibits poor prognosis and metastases are the main cause of death. The discovery of reliable prognostic indicators of tumour progression could greatly improve clinical practice. MicroRNAs are involved in the regulation of basic cellular processes such as cell proliferation, differentiation, and apoptosis. Since miRNAs have been shown to be abnormally expressed in different tumours their importance as potential cancer prognostic indicators is increasing. To define the role of miRNA in OSCC tumours we investigated the expression profile of 15 OSCC (8 without metastasis and 7 with lymph node metastasis) using microarray analysis. Thirteen miRNA were significantly overexpressed (miR-489, miR-129, miR-23a, miR-214, miR-23b, miR-92, miR-25, miR-210, miR-212, miR-515, miR-146b, miR-21, miR-338) and 6 miRNA were underexpressed (miR-520h, miR-197, miR-378, miR-135b, miR-224, miR-34a) in oral tumours. Underexpression of mir-155, let-7i, mir-146a was found to characterize progression to metastastatic tumours. Further investigations will elucidate whether differentially expressed miRNAs will help to better classify OSCCs, thus improving diagnoses and patient care.

Survivin expression in oral squamous cell carcinoma

A series of 110 cases of oral squamous cell carcinoma (SCC) together with six lymph node and one distant metastatic lesions was analysed for expression of survivin, a recent apoptosis inhibitor, by immunohistochemistry and Western blotting. In total, 91 cases (82.7%) of carcinoma and all metastasis (seven cases, 100%) were positive for survivin expression, with weighted survivin scores ranging from 1 to 4. In contrast, normal oral epithelium did not express survivin. There was no significant correlation between survivin expression and age, sex, tumour size, the presence of lymph node and distant metastases. Survivin expression was increased in poorly differentiated tumours, even if differences were not statistically significant. In contrast, when analysed for prognostic significance, patients with low survivin expression had statistically significant better survival rates than the group with high survivin expression (P<0.05). These data suggest that survivin expression may identify cases of oral SCC with more aggressive and invasive phenotype.

Expression of proliferating cell nuclear antigen in ameloblastomas and odontogenic cysts

The identification of the proliferative activity in tumours may be useful to predict the biological behaviour of different lesions. Proliferating cell nuclear antigen (PCNA) has been used for the evaluation of the proliferative ability of many lesions. In this study 22 ameloblastomas (4 follicular, 5 plexiform, 4 acanthomatous, 5 unicystic, 4 recurrent), 12 odontogenic keratocysts (OKC), 8 dentigerous cysts (DC), and 12 radicular cysts (RC) were analysed. PCNA+ cells were present in all cyst types but the OKC contained the highest number of PCNA+ cells. In OKC the location of PCNA+ cells was mainly suprabasal. In ameloblastoma PCNA+ cells were located mainly in the peripheral portion of the tumour islands. Statistical analysis showed that ameloblastoma had higher PCNA+ cell counts than OKC (P < 0.0001); OKC had higher values than DC and RC (P < 0.0001). Recurrent ameloblastoma presented higher PCNA+ cell counts than other types of ameloblastoma, while unicystic ameloblastoma showed lower values than acanthomatous, plexiform and follicular ameloblastomas (in this latter case the difference was not statistically significant). These data could help to explain the different biological behaviour of these lesions.

Vascular Endothelial Growth Factor (VEGF) Expression in Healthy and Inflamed Human Dental Pulps

Vascular endothelial growth factor (VEGF) is a glycoprotein that has the capability to increase vascular proliferation and permeability. VEGF has been found to be expressed in several different types of tumors, and it may contribute to the progression of malignant tumors. Immunostaining for VEGF and factor VIII was performed in normal healthy pulps and in irreversible pulpitis. In both cases the vessels were always positive for VEGF. Our immunohistochemical data show that the expression of VEGF was strongly positive in the inflammatory infiltrate in irreversible pulpitis. VEGF expression in the stromal cells in healthy pulps ranged from 20 to 100% (with a mean of 68.82), and in irreversible pulpitis ranged from 0 to 100% (with a mean of 62.35%); this difference was statistically significant (p = 0.05). This down-regulation in the stromal cells in irreversible pulpitis could be due to the presence, in a low compliance system such as the dental pulp, of inflammatory infiltrate. VEGF is probably a factor implicated in the etiology and progression of pulpitis. The microvessel density in healthy pulps was 90.00 +/- 27.5, while, in irreversible pulpitis, it was 56.68 +/- 21.15. This difference was statistically significant (p = 0.001). The decrease in microvessel density in irreversible pulpitis could be related to failing vascular function and blood flow decrease.

Effect of c-Met Expression on Survival in Head and Neck Squamous Cell Carcinoma

The proto-oncogene c-Met has been suggested to be associated with progression of squamous cell carcinoma of the head and neck. The aims of the present study were to assess the prevalence of c-Met expression in oral squamous cell carcinoma (OSCC) and to verify whether c-Met can be considered a marker of prognosis in these patients. In a retrospective study, a cohort of 84 OSCC patients was investigated for c-Met expression and its cellular localization by immunohistochemistry. After grouping for c-Met expression, OSCC patients were statistically analyzed for the variables age, gender, histological grading, tumor node metastasis, staging and overall survival rate. Univariate and multivariate statistics were used for data analysis. Sixty-nine cases (82.2%) of OSCC showed immunopositivity, with a mainly membranous expression and scattered areas also showing a cytoplasmic localization, whereas 15 cases (17.8%) did not show c-Met. No statistical association was found between c-Met expression and any variables considered at baseline, apart from the higher number of c-Met positivity in females (p = 0.026). Among positive tumors, well-differentiated areas showed low or absent cytoplasmic expression, while low-differentiated areas showed both membranous and cytoplasmic positivity. In terms of prognostic significance, c-Met expression was found to have an independent association with a poorer overall survival rate (p = 0.036). On the basis of these results, it is possible to suggest c-Met as an early marker of poor prognosis, a hallmark of aggressive biological behavior in OSCC, suggested to be useful in identifying cases of OSCC before the relapse.

Bone regeneration with calcium sulfate: evidence for increased angiogenesis in rabbits.

Autologous bone is the preferred bone graft material because it carries proteins as bone-enhancing substrates, minerals, and vital bone cells. Calcium sulfate (CS) is a well-tolerated, biodegradable, osteoconductive bone graft substitute and is a reasonable alternative to autogenous bone graft. Blood vessels are an important component of bone formation and maintenance. The process of vascular induction is called angiogenesis, and it plays a key role in all regenerative processes. Bone tissue differentiation is related to the local presence of blood vessels. One method to evaluate the presence of blood vessels in a tissue is to count the microvessels to evaluate microvessel density (MVD). The aim of the present study was to conduct a comparative evaluation of microvessel density in sites treated with CS and autologous bone in rabbits, with or without e-PTFE nonresorbable membranes (Gore-Tex, Flagstaff, Ariz). Nine New Zealand rabbits, each weighing about 2.5 kg, were used in this experiment. Three 6-mm wide defects were created in each tibial metaphysis. The defects were filled in a random way. The defects of group 1 (3 rabbits) were filled with CS granules (Surgiplaster, Classimplant, Rome, Italy) and covered with e-PTFE membranes. The defects in group 2 (3 rabbits) were filled with CS granules (Surgiplaster). The defects in group 3 (3 rabbits) were filled with autologous bone. A total of 54 defects were filled (18 with CS and e-PTFE membranes, 18 with CS alone, and 18 with autologous bone). No postoperative deaths or complications occurred. All nine animals were sacrificed at 4 weeks. MVD results were as follows: in the first group, 9.88 +/- 4.613; in the second group, 7.92 +/- 1.998; and in the third group, 5.56 +/- 1.895. P = .000 was highly significant. Statistically significant differences were found between groups 1 and 3, 1 and 2, and 2 and 3. The presence of more blood vessels in the sites treated with CS could help to explain the good results reported in the literature with the use of CS.

p53 Protein Expression in Odontogenic Cysts

p53 protein seems to be related to the suppression of cell proliferation. p53-positive tissues seem to have a higher proliferative activity than p53-negative ones. Odontogenic keratocyst (OKC) has a different behavior from other types of cysts because it is more aggressive, with a tendency to recurrence. Twenty-two dentigerous cysts, 24 radicular cysts, and 20 OKCs were used in the present study. Two dentigerous cysts (9.1%), 2 radicular cysts (8.3%), and 9 OKCs (45%) expressed the p53 protein. The differences between the three groups were statistically significant (p = 0.003). In 10 cases of OKCs epithelial dysplasia was found. One of the 10 OKCs without dysplasia and 8 of the 10 OKCs with dysplasia were p53-positive: the difference between the two groups was statistically significant (p = 0.007). The overexpression of p53 protein was not on the other hand correlated with the occurrence of multiple, bilateral, and recurrent OKCs. Moreover the distribution of p53-positive cells was parabasal in contrast with other types of cysts. These qualitative and quantitative differences in proliferative activity in OKCs seem to point to an alteration in cell cycle control.

Osteolipoma of the tongue

Lipomas are common, benign tumours located in any part of the body in which fat is normally present. Some variants of lipoma have been described according to the type of tissue present. A rare variant consists of a lipoma with osseous or cartilaginous metaplasia. These lesions have been called chondrolipoma, osteolipoma, lipoma with chondroid or osseous metaplasia, lipoma with cartilaginous or osseous change, or ossifying lipoma. We present the case of an osteolipoma of the tongue in a 49-year-old female who was referred for a painless mass on the left lateral margin of the tongue, and present for about 8 years. Osteolipomas have been reported in middle-aged or elderly patients with a very long clinical history. These tumours tend to be large and to arise from the deep soft or subcutaneous tissues. The cartilage and bone is probably produced by metaplasia of fibroblasts in chondroblasts or osteoblasts. These lesions are benign and do not recur.

Potential markers of tongue tumor progression selected by cDNA microarray.

Squamous cell carcinoma (SCC), the most frequent malignant tumor of the oral cavity, generally exhibits a poor prognosis and metastases are the main cause of death. This tumor often arises from pre-malignant lesions. To date, it is difficult to predict if and which pre-malignant lesions may progress into oral SCC using traditional methods. For these reasons, several studies are trying to identify markers useful in the progression of pre-malignant lesions and tumors. To define the genetic expression profile of tongue tumor progression we compared 9 dysplasias (DS), 8 tumors without metastasis (TWM), 11 metastasizing SCCs (MT) of the tongue, and a baseline of 11 normal tissues by using cDNA microarray containing 19.2 K clones. We initially applied hierarchical agglomerative clustering based on information from all 6026 clones. Results were obtained by performing a two steps analysis: a Significance Analysis of Microarray (SAM) and a Gene Ontology search. One hundred and five clones have statistically significant different expression levels (FDR <0.01) between DS and TWM, whereas 570 genes have statistically significant difference expression levels between TWM and MT (FDR <0.01) as detected by SAM. By filtering with FatiGo only 33 genes were differentially expressed in TWN, respect to DS, whereas 155 genes were differentially expressed in MT respect to TWM. We detected some genes which encode for oncogenes, transcription factors and cell cycle regulators as potential markers of DS progression. Examples are BAG4, PAX3 and CCNI, respectively. Among potential markers of metastases are some genes related to cell mobility (TSPAN-2 and SNTA1), intercellular adhesion (integrin alpha 7) or extracellular matrix components (ADAMTS2 and cathepsin O). Additionally, under-expressed genes encoded apoptosis-related proteins (PDCD4 and CASP4). In conclusion, we identified several genes differentially expressed in tumor progression which can potentially help in better classifying premalignant lesions and tongue SCCs.

bcl-2 expression and apoptotic bodies in 13-cis-retinoic acid (isotretinoin)-topically treated oral leukoplakia: a pilot study.

In a double-blind study 10 patients with oral leukoplakia were treated daily (three topical applications) with 0.1% isotretinoin gel or a placebo for 4 months. Nine patients completed the treatment, while one patient was lost to follow-up. Subsequently, the patients who had received the placebo used the active medication for an additional 4 months. All patients treated with the active medication showed a significant improvement of the oral lesions while, in the patients receiving only the placebo, the size of the lesions remained the same. Also the group of patients who received the active medication after the placebo showed an improvement in the size and clinical appearance of the lesions. A complete response was defined as the complete disappearance of the lesion as assessed by visual inspection, while a partial response was defined as a 50% or more reduction in the size of the lesions. In total we had a complete response and eight partial responses. No side-effects from the use of the gel were ever observed. The percentage of bcl-2-positive cells was evaluated in the basal layer from a minimum of 1000 cells in each case and the bcl-2 immunostaining was scored using three groups: - (< or = 10% cells); + (< or = 50% cells); +2 (> or = 50% cells). The presence of apoptotic bodies was evaluated in a random fashion in the parabasal layer in 20 HPF. Immunohistochemical analysis for bcl-2 protein showed that before treatment a weak positivity of the basal layers, with a focal positivity of some parabasal cells, was present: five out of nine specimens were positive. Only a few apoptotic bodies were observed. After treatment in almost all specimens it was possible to observe a complete bcl-2 negativity with a positivity in only one specimen out of nine. An increase in apoptotic bodies was observed. Statistical analysis showed that the difference between the bcl-2 positivity in the two groups was not statistically significant (P = 0.134) while, on the contrary, the difference in the count of the apoptotic bodies between the same two groups was statistically significant (P = 0.0193). In conclusion, the data obtained from this pilot study show that good results can be obtained with the topical use of 13-cis-retinoic acid.