ABSTRACT
OBJECTIVE: The aim of this study was to evaluate the DNA methylation profile in 22 apoptosis-related genes in pleomorphic adenomas (PAs) of the salivary glands, in comparison with normal salivary glands (NSGs), and to address the differences in methylation patterns between smaller and larger tumors. Additionally, we investigated if the hypermethylation of differentially methylated genes between NSGs and PAs impacted the messenger RNA (mRNA) transcription. DESIGN: Twenty-three fresh PA samples and 12 NSG samples were included. The PA samples were divided into 2 groups: PAs with clinical size larger than 2 cm (n = 12) and PAs with clinical size 2 cm or smaller (n = 11). DNA methylation at the promoter region of a panel of 22 genes involved in apoptosis was profiled by using a human apoptosis DNA methylation polymerase chain reaction array, and the transcriptional levels of genes showing differential methylation profiles between PAs and NSGs were assessed. RESULTS: TNFRSF25 and BCL2 L11 were highly methylated in PAs, in comparison with NSGs, irrespective of tumor size. However, no difference could be observed in the mRNA transcription between PAs and NSGs. CONCLUSIONS: Hypermethylation of the proapoptotic genes BCL2 L11 and TNFRSF25 is observed in PA. However, this phenomenon did not impact mRNA transcription.
Subject(s)
Adenoma, Pleomorphic/genetics , Bcl-2-Like Protein 11/genetics , DNA Methylation , Polymerase Chain Reaction , Receptors, Tumor Necrosis Factor, Member 25/genetics , Salivary Gland Neoplasms/genetics , Adenoma, Pleomorphic/pathology , Adult , Aged , Apoptosis , Female , Humans , Male , Middle Aged , Salivary Gland Neoplasms/pathologyABSTRACT
OBJECTIVE: To assess intratumor molecular heterogeneity in salivary gland pleomorphic adenoma (PA) and to investigate if intratumor molecular heterogeneity is associated with cell proliferation or apoptotic indexes. STUDY DESIGN: Nine formalin-fixed paraffin-embedded samples of PA of salivary glands were included in the study. Cell proliferation was estimated by using Ki-67 immunohistochemistry, and apoptotic index was achieved by combining terminal deoxynucleotidyl transferase dUTP nick end-labeling with morphology. A minimum of two different tumor areas of each sample was microdissected, and DNA was extracted. DNA extracted from the tumor capsule was used as normal matched control. Different tumor areas were at least 4 mm from one another and comprised tumor cell-rich areas. Loss of heterozygosity was assessed by a panel of six polymorphic microsatellite markers located at chromosomes 3 (D3 S1029), 9 (D9 S162 and D9 S157), 11 (D11 S1369), and 17 (P53 and AFM238 WF2). RESULTS: Six of nine samples showed intratumor heterogeneity on the basis of the loss of heterozygosity findings. Intratumor molecular heterogeneity did not show association with cell proliferation or apoptotic indexes (P > .05). CONCLUSIONS: Our findings point to the existence of intratumor molecular heterogeneity in salivary gland PA. This is an advance in the efforts to clarify PA molecular pathogenesis, showing that this characteristic is not exclusive to malignant solid tumors.
Subject(s)
Adenoma, Pleomorphic/genetics , Adenoma, Pleomorphic/pathology , Salivary Gland Neoplasms/genetics , Salivary Gland Neoplasms/pathology , Adult , Aged , Apoptosis , Cell Proliferation , Chromosomes, Human, Pair 11 , Chromosomes, Human, Pair 17 , Chromosomes, Human, Pair 3 , Chromosomes, Human, Pair 9 , DNA, Neoplasm/analysis , Female , Humans , Immunohistochemistry , In Situ Nick-End Labeling , Loss of Heterozygosity , Male , Middle Aged , Polymerase Chain ReactionABSTRACT
BACKGROUND: Ameloblastoma is a locally invasive neoplasm often associated with morbidity and facial deformities, showing increased Epidermal Growth Factor Receptor (EGFR) expression. Inhibition of EGFR was suggested as a treatment option for a subset of ameloblastomas. However, there are resistance mechanisms that impair anti-EGFR therapies. One important resistance mechanism for EGFR-inhibition is the EGFR nuclear localization, which activates genes responsible for its mitogenic effects, such as Cyclin D1. METHODS: We assessed EGFR nuclear localization in encapsulated (unicystic, n = 3) and infiltrative (multicystic, n = 11) ameloblastomas and its colocalization with Cyclin D1 by using anti-EGFR and anti-lamin B1 double labeling immunofluorescence analyzed by confocal microscopy. Oral inflammatory fibrous hyperplasia and oral squamous cell carcinoma samples were used for comparison. RESULTS: Twelve cases of ameloblastoma exhibited nuclear EGFR colocalization with lamin B1. This positive staining was mainly observed in the ameloblast-like cells. The EGFR nuclear localization was also observed in control samples. In addition, nuclear EGFR colocalized with Cyclin D1 in ameloblastomas. CONCLUSIONS: Nuclear EGFR occurs in ameloblastomas in association with Cyclin D1 expression, which is important in terms of tumor biology clarification and raises a concern about anti-EGFR treatment resistance in ameloblastomas.
