Methylation analysis of Rb, P16, MGMT, and hMLH-1in mucoepidermoid carcinomas. A pilot study

Mucoepidermoid carcinoma (MC) is the most common malignant epithelial neoplasm in the salivary glands. This neoplasm has varying proportions of mucous, epidermoid, intermediate, columnar, and clear cells. MCs have been associated with CRTC1-MAML2 genes; however, their pathogenesis is uncertain. Recently, epigenetic changes have been considered a possible aetiologic factor. To identify the methylation state of RB, P16, MGMT, and hMLH genes in the three severity grades of MC were used five MCs and one healthy minor salivary gland as a control group (CG) obtained from the Pathology and Oral Medicine Laboratory and analyzed using MS-PCR to compare the presence or absence of methylation in promotor regions. The Kruskal- Wallis test was performed, with p≤0.05 considered significant. CG was employed as the normalizer of methylation levels. All assays were performed in triplicate. The mean age of our population was 52.6±18.6 years old; the total population was female and included 2 low grade, 2 intermediate grade, and 1 high grade levels of severity. When comparing the methylation status of the three histopathological grades of MC against the control, statistically significant differences were observed in Rb-M, MGMT-M, and hMLH-1-NM for high-grade severity, with p values of 0.03, 0.05, and 0.04, respectively. Methylation is a possible mechanism for pathogenesis processing of high-grade MC. However, a larger sample population is necessary to validate this finding.

El carcinoma mucoepidermoide (CM) es la neoplasia epitelial maligna más frecuente de glándulas salivales. Esta neoplasia tiene proporciones variables de células mucosas, epidermoides, intermedias, cilíndricas y claras. Los CM se han asociado con los genes CRTC1-MAML2; sin embargo, su patogenia es incierta. Recientemente, los cambios epigenéticos se han considerado un posible factor etiológico. Para identificar el estado de metilación de los genes RB, P16, MGMT y hMLH en los tres grados de severidad de CM se utilizaron cinco CM y una glándula salival menor sana como grupo control (GC) obtenidos del Laboratorio de Patología y Medicina Oral y analizados mediante MS-PCR para comparar la presencia o ausencia de metilación en regiones promotoras. Se realizó la prueba de Kruskal-Wallis, considerándose significativa una p≤0,05. Se empleó GC como normalizador de los niveles de metilación. Todos los ensayos se realizaron por triplicado. La edad media de nuestra población fue de 52,6 ± 18,6 años; la población total era femenina e incluía 2 niveles de severidad de grado bajo, 2 de grado intermedio y 1 de alto grado. Al comparar el estado de metilación de los tres grados histopatológicos de CM contra el GC, se observaron diferencias estadísticamente significativas en Rb-M, MGMT-M y hMLH-1-NM para severidad de alto grado, con valores de p de 0.03, 0.05, y 0,04, respectivamente. La metilación es un posible mecanismo para el procesamiento de patogénesis de CM de alto grado. Sin embargo, se necesita una población de muestra más grande para validar este hallazgo.

Multiple approaches to oral epithelial dysplasia degree analyses: a pilot study.

BACKGROUND: Oral epithelial dysplasia (OED) is the presence of cells of an abnormal type within a tissue, which may signify a stage preceding the development of cancer. Our aim was to determine the interrelation between the expression of multiple molecular markers and the histological features of oral dysplasia. METHODS: Fifteen samples of OED (five for each severity degree) were analyzed through software assisted image cytometry nuclear morphology. p53 (wild-type and mutated form), Bax and Bcl2 expression was immunohistochemically determined, and the gene expression of MMP1, MMP2, MMP9 and hTERT was determined by RT-PCR. The mean, standard deviation, ANOVA and Fisher's Exact Test (P<0.05) were performed. RESULTS: Our analysis indicated congruence between the software-assisted measurement of nuclear morphology and severity degree. Only five samples were positive to p53-mutated form; and Bax was more expressed than Bcl-2. hTERT expression was significantly expressed in relation to severity, and MMP1 was predominantly expressed, followed by MMP9 and MMP2. CONCLUSIONS: Our results reinforce that software-assisted measurement is an alternative to severity degree determination. MMP1 is an important marker for severity dysplasia degree; however, the predominant expression of Bax over Bcl-2 suggests that this pro-apoptotic state could be used to minorize the progression, perhaps, as a future therapeutic target.

Ectodermal and ectomesenchymal marker expression in primary cell lines of complex and compound odontomas: a pilot study.

BACKGROUND: Odontomas are odontogenic tumors with hamartoma features that are classified as compound or complex. Our objective was to characterize the proliferation of ectodermal and ectomesenchymal profile markers of primary cell cultures of complex and compound odontomas. METHODS: Four samples of compound odontomas (OdCm) and three of complex odontomas (OdCx) were obtained from patients attending the Oral Pathology and Medicine Clinic of the Graduate Dental School, National Autonomous University of Mexico for primary culture generation. MTT, immunocytochemistry and RT-PCR assays of CD34, Sox2, Amel, Ambn, p21, EDAR, Msx1, Msx2, Pax9, RUNX2, BSP, OPN, Barx1 and GAPDH (control) were performed. Additionally, six paraffin-embedded odontomas were obtained for immunocytochemistry and RT-PCR validation assays. The mean and standard deviation were determined, and ANOVA and Kruskall-Wallis tests were performed. RESULTS: Cultured compound odontoma exhibited higher proliferation, and an ectomesenchymal immunocytochemistry profile with predominant expression of Amel, BSP, Pax9, EDAR, Barx and Msx2; in complex cultured odontoma Sox2, CD34, RUNX2 and OPN predominated. Our statistical analysis showed a significant difference in PCR analysis (P<0.05) for OPN and CD34. Paraffin-embedded odontomas showed similar pattern with difference for NGFR and Sox2 for immunohistochemistry and EDAR, BARX1 and PAX9 for RT-PCR assays. CONCLUSIONS: The results suggested heterogeneous behavior for both odontoma cell lines, because in compound odontomas predominant biomarkers are related to the enamel knot, late-stage odontogenesis and ectomesenchymal interactions; and in complex odontoma the significant expression of CD34 and OPN could be responsible for the difference behavior and mineralized amorphous structure.