Intratumor molecular heterogeneity in pleomorphic adenoma of the salivary glands.

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.

BRAF V600E and loss of heterozygosity assessment in benign oralneural tumours.

BACKGROUND: The purpose of this study was to evaluate loss of heterozygosity (LOH) and to assess BRAF V600E mutation in oral neurofibromas, palisaded encapsulated neuromas (PEN) and schwannomas. METHODS: Six oral neurofibroma, 5 PEN and 3 schwannoma samples were included in the study. LOH was assessed using polymorphic microsatellite markers at chromosome regions 3p (marker D3S1029), 9p (markers D9S171, D9S162, D9S157), 11q (marker D11S1369), and 17p (markers AFM238WF2 and P53), and results were evaluated after capillary electrophoresis. BRAF mutation encoding V600E was assessed by real-time PCR with a specific TaqMan probe to detect the T>A transversion at position c.1799. RESULTS: LOH occurred at chromosomes 3p (marker D3S1029), 11q (D11S1369) and 17p (AFM238WF2 and P53). LOH occurred in 2/6 neurofibromas, 2/5 PEN and in none of the 3 schwannoma samples. The 6 neurofibromas, 2/2 PEN evaluated and the 3 schwannomas were BRAF wild type. CONCLUSION: According to our results, oral benign peripheral nerve sheath tumours have a low LOH rate, but P53 locus alteration is occasionally found. Additionally, BRAF V600E mutation is either not relevant to the molecular pathogenesis of this group of lesions of the oral cavity, or may occur at very low rates.

Inter- and intra-lesional molecular heterogeneity of oral leukoplakia.

OBJECTIVES: Oral leukoplakia (OL) is the most common potentially malignant lesion of the oral cavity, and OL diagnosis is a risk factor for developing subsequent oral squamous cell carcinomas (OSCC). Notably, loss of heterozygosity (LOH) profiles have been validated as risk predictors of malignant transformation of OL. Similar to other solid malignant tumors, OSCC exhibit molecular heterogeneity. However, if and to what extent tumor heterogeneity is present in premalignant lesions of the oral cavity has not been addressed. As LOH analysis is currently being used to stratify OL patients at risk for OSCC development in chemoprevention studies, insight into the issue of molecular heterogeneity of OL is of clinical significance. MATERIALS AND METHODS: To address this issue, 11 polymorphic microsatellite markers localizing to chromosomes 3, 9, 11 and 17 were used to detect LOH in 28 samples of 14 OL patients, by capillary electrophoresis analysis. These samples were either clinically recurrent lesions, or two anatomically distinct biopsies from the same lesion, or even two different OL lesions located at distinct intraoral sites. RESULTS: In all but one of the biopsies pairs, distinct LOH patterns were displayed regardless of histopathological grade. These data provide evidence for inter- and intra-lesional molecular heterogeneity in OL. CONCLUSIONS: On the basis of these findings, molecular heterogeneity needs to be addressed in subsequent studies targeting specific carcinogenic pathways/genes in chemoprevention of malignant transformation of OL.

Loss of heterozygosity of the PTCH gene in ameloblastoma.

Ameloblastoma is a locally aggressive benign neoplasm derived from odontogenic epithelium, with high recurrence rates. Alterations in the Sonic Hedgehog signaling pathway, including PTCH gene mutations, have been associated with the pathogenesis of some odontogenic tumors. The purpose of the present study was to assess loss of heterozygosity at the PTCH locus in ameloblastoma. Twelve ameloblastomas were included, and loss of heterozygosity was assessed by using 3 microsatellite markers D9S252, D9S127, and D9S287 and 3 single-nucleotide polymorphisms rs112794371, rs111446700, and rs357564, all located at the PTCH gene locus. Furthermore, we investigated GLI1 and GLI2 transcription levels by quantitative reverse transcription polymerase chain reaction in 8 ameloblastomas and, concomitantly, PTCH protein levels by immunohistochemical analysis. Loss of heterozygosity at 9q21.33-9q.31 was detected in 4 (40.0%) of 10 informative cases of ameloblastoma. All 8 analyzed samples expressed GLI1 messenger RNA and 7 cases GLI2 messenger RNA. Interestingly, loss of heterozygosity at the PTCH locus was not correlated with GLI1 or GLI2 transcription levels, nor was there any correlation with PTCH protein expression. In conclusion, our findings suggest that loss of heterozygosity in the PTCH region may be relevant to the pathogenesis of ameloblastoma but may target a different gene than PTCH.

Evidence of loss of heterozygosity of the PTCH gene in orthokeratinized odontogenic cyst.

The orthokeratinized odontogenic cyst (OOC) is an odontogenic cyst of unknown etiology. Clinical, histological, and biological differences are reported between keratocystic odontogenic tumor (KOT) and OOC. PTCH is a tumor suppressor gene related to sonic hedgehog (SHH) pathway important in embryological development. Considering that alterations in this pathway have been described in sporadic and nevoid basal cell syndrome-associated KOT, we tested the hypothesis that OOC is also associated with loss of heterozygosity (LOH) of the PTCH gene. Seven samples of OOC and seven of KOT were included in the study. D9S287, D9S196, and D9S127 microsatellite markers located in the region of PTCH gene, at chromosome 9q, were investigated for LOH. There was loss in at least one locus in 5/7 KOT and in 4/7 OOC samples. The present finding demonstrates that, despite the existence of clinical, morphological, immunohistochemical, and biological behavior differences between OOC and KOT, both harbor similar genetic alterations at 9q.