Fabrício Rezende Amaral

Novel mutations in the SH3BP2 gene associated with sporadic central giant cell lesions and cherubism.

Central giant cell lesion (CGCL) is a reactive bone lesion that occurs mainly in the mandible, characterized by the multinucleated osteoclast-like giant cells in a background of oval to spindle-shaped mononuclear cells. The etiology is unknown and occurs more commonly in young adults. Cherubism, a rare disease found predominantly in females has histologic characteristics indistinguishable from those of CGCL and is caused by mutations mostly present in exon 9 of the SH3BP2 gene. In this study, we investigated four cases of CGCL and one case of cherubism. DNA was extracted from peripheral blood and tumor tissue and all coding and flanking regions of the SH3BP2 amplified by PCR and directly sequenced to identify underlying mutations. Two novel mutations were found; a heterozygous missense mutation c.1442A>T (Q481L) in exon 11 in one sporadic case of CGCL and a heterozygous germline and tumor tissue missense mutation c.320C>T (T107M) in exon 4 in one patient with cherubism. These findings open a new window to investigate the possible relationship between the pathogenesis of the cherubism and CGCL.

Cell proliferation and apoptosis in ameloblastomas and keratocystic odontogenic tumors

A high proliferative activity of the odontogenic epithelium in ameloblastoma (AM) and keratocystic odontogenic tumor (KOT) has been demonstrated. However, no previous study has simultaneously evaluated cell proliferation and apoptotic indexes in AM and KOT, comparing both lesions. The aim of this study was to assess and compare cell proliferation and apoptotic rates between these two tumors. Specimens of 11 solid AM and 11 sporadic KOT were evaluated. The proliferation index (PI) was assessed by immunohistochemical detection of Ki-67 and the apoptotic index (AI) by methyl green-pyronine and in situ DNA nick end-labelling methods. KOT presented a higher PI than AM (p<0.05). No statistically significant difference was found in the AI between AM and KOT. PI and AI were higher in the peripheral cells of AM and respectively in the suprabasal and superficial layers of KOT. In conclusion, KOT showed a higher cell proliferation than AM and the AI was similar between these tumors. These findings reinforce the classification of KOT as an odontogenic tumor and should contribute to its aggressive clinical behavior.

The highly prevalent H3F3A mutation in giant cell tumours of bone is not shared by sporadic central giant cell lesion of the jaws

OBJECTIVE Central giant cell lesion (CGCL) and giant cell tumour (GCT) are bone lesions that share similar microscopic features. Recently, it was reported that 90% of bone GCT exhibit either p.Gly34 Trp or p.Gly34 Leu in H3F3A, one of two genes for histone H3.3 located on chromosome 1. We aimed to test whether sporadic CGCL of the jaws share the H3F3A mutations reported in GCT of other bones. METHODS Nine samples of CGCL of the jaws were included in the study, and mutations were assessed by direct sequencing. RESULTS None of the CGCL samples presented the recurrent p.Gly34 Trp or p.Gly34 Leu mutations in the H3F3A gene. CONCLUSION On the basis of our findings, H3F3A p.Gly34 Trp or p.Gly34 Leu mutations are not a frequent event in CGCL. If these alterations are confirmed to be exclusive of GCT, the assessment of H3F3A mutations may help in the differential diagnosis of GCT and CGCL of the jaws.

NFATc1 and TNFα expression in giant cell lesions of the jaws

BACKGROUND Activation mutations of SH3BP2 gene have been demonstrated in cherubism and central giant cell lesion (CGCL). In the present study we first attempted to investigate the SH3BP2 gene in peripheral giant cell lesion (PGCL). The effect of SH3BP2 gene mutations on the transcription of the downstream genes nuclear factor of activated T cells (NFATc1) and the cytokine tumor necrosis factor-alpha (TNF-alpha) was also investigated together with the immunolocalization of NFATc1 protein in a set of cases of PGCL, CGCL and cherubism with and without SH3BP2 mutation. METHOD Fresh samples of five PGCL, five CGCL and one cherubism cases were included in this study. One of the samples of CGCL presented a somatic heterozygous mutation c.1442A>T in exon 11. The cherubism case showed a heterozygotic substitution c.320C>T in both blood and lesion. These mutations were previously published. All coding and flanking regions of the SH3BP2 gene were sequenced in the cases of PGCL. The real-time polymerase chain reaction (RT-PCR) was performed to analyze the transcription of NFATc1 and TNF-alpha genes. The immunohistochemical analysis of the NFATc1 protein was also performed. RESULTS No SH3BP2 gene mutation was found in PGCL. The RT-PCR showed increased expression of NFATc1 and decreased transcription of TNF-alpha in all the samples. The immunohistochemical analysis of the NFATc1 protein showed a predominant nuclear staining in the multinucleated giant cells. CONCLUSION The development of giant cells lesions of the jaws and cherubism are possibly mediated by overexpression of NFAT in the nucleus of the multinucleated cells.

Quantitative expression analysis of apoptotic/antiapoptotic genes and association with immunolocalization of BAX and BCL-2 in peripheral and central giant cell lesions of the jaws

Central giant cell lesion (CGCL) and peripheral giant cell lesion (PGCL) of the jaws are characterized by multinucleated osteoclast-like giant cells in a background of mononuclear cells. While mononuclear cells retain proliferative activity in both lesions, giant cells are Ki-67 negative. This observation raised the theory that giant cells are formed by cytoplasmic fusion of mononuclear cells, and also that these lesions are of reactive nature. As the giant cells are not proliferating in CGCL and PGCL, apoptosis of such cells should be investigated. We investigated the transcription of BAX and BCL-2 mRNAs in six fresh samples of CGCL and six fresh samples of PGCL by qRT-PCR (quantitative reverse transcription PCR) and used immunohistochemistry to demonstrate the localization of these proteins, as well as caspase 3 active in six paraffin-embedded samples of CGCL and nine paraffin-embedded samples of PGCL. While both groups showed increased expression of BAX and BCL-2 mRNA, PGCL showed a higher apoptotic index (ratio BAX/BCL-2) than CGCL. The three proteins investigated were expressed almost exclusively in the cytoplasm of giant cells. To further confirm apoptotic activity, we performed TUNEL analysis in the same samples of the immunohistochemistry and found a higher positivity in the giant cells of PGCL compared to the giant cells of CGCL. Our results show increased expression of apoptotic-related genes in both PGCL and CGCL and that the giant cells are probably the main source of these events. Also, it raises a hypothesis that differences in the apoptotic activity might be associated with the different clinical behavior of CGCL and PGCL.

Clonality analysis of giant cell lesions of the jaws

Despite the importance of clonality to understand the pathogenesis and progression of tumors, it has not been investigated yet in giant cell lesions of the jaws. The aim of this study was to analyze the clonality of peripheral giant cell lesions (PGCL) and central giant cell lesions (CGCL) of the jaws. Six samples of PGCL and 5 samples of CGCL were analyzed in this study using the polymorphic human androgen receptor locus (HUMARA) assay. Three out of the 5 samples of the CGCL and 3 out of 6 samples of PGCL exhibited a monoclonal pattern. Our findings demonstrate that some giant cell lesions of the jaws are clonal, which indicate that these lesions may have a common genetic mechanism of development. Further studies are necessary to better elucidate the molecular mechanisms involved in the pathogenesis of such lesions.

WWOX expression in giant cell lesions of the jaws

OBJECTIVE To investigate WWOX messenger RNA (mRNA) transcriptional levels in giant cell lesions (GCLs) of the jaws and associate its expression with clinical parameters. STUDY DESIGN In this pilot study, quantitative reverse-transcription polymerase chain reaction was performed to analyze WWOX expression in 6 central giant cell lesions (CGCLs) (including 2 aggressive), 5 peripheral giant cell lesions (PGCLs) and 1 cherubism sample. Immunohistochemistry was performed to confirm the localization of the Wwox protein. RESULTS CGCL and PGCL showed an overall increased expression of WWOX, as did the cherubism case, but no differences were observed among the groups. Wwox was localized almost entirely to the cytoplasm of multinucleated giant cells, as well as in a few mononuclear cells. CGCL and PGCL showed higher expression of the WWOX mRNA than peripheral blood mononuclear cells. The 2 aggressive CGCL samples exhibited decreased WWOX expression. CONCLUSIONS These results showed increased expression of WWOX mainly in non-aggressive GCLs of the jaws.

Oral giant cell granuloma in a patient with glycogen storage disease.

The glycogen storage disease (GSD) is a group of inherited disorders that involve deficiencies in the enzymes that metabolize glycogen. The purpose of the present paper is to report a rare case of GSD type 1b that presented both peripheral and central giant cell granuloma, and to discuss the possible explanation for this unusual finding. The use of corticosteroids in the management of central giant cell granuloma is also demonstrated.

Abstract 2728: Clonal nature of benign and malignant salivary gland neoplasms

The pathogenesis of salivary gland neoplasms (SGN) has not been established. There is a real debate on clonality issues and all the definitive evidence is on the side of polyclonal tumor origin. Evidence that many tumor types may be polyclonal in origin has been published. Despite all this body of evidence, scientists have incorporated the clonal theory proposed by Knudson (1985) and only strengthen these results (and do not discuss contrary findings), because they think it is expected. There are few clonality studies on benign salivary neoplasms and almost no data on malignant neoplasms. The HUMARA assay was used in the present study to investigate the clonality of 27 samples of SGN (17 benign and ten malignant). Briefly, samples were digested with HhaI and HpaII methylation-sensitive enzymes and a PCR carried out. Eighteen out of 27 cases revealed monotypy, while three were non-informative and six showed heterotypy, indicating a true polyclonality. In summary, our results show evidence that a subset of SGN might be clonal in origin, while some samples are polyclonal. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2728. doi:10.1158/1538-7445.AM2011-2728