Materials and methods

Formalin-fixed paraffin-embedded blocks from 21 consecutive cases of primary CGCLs from patients who underwent treatment with intralesional triamcinolone hexacetonide injections (20 mg/ml diluted in an anaesthetic solution of 2% lidocaine/epinephrine 1:200.000 in a ratio of 1:1; 1.0 ml of the solution infiltrated every 1 cm ³ of radiolucide area of the lesion totalling 6 biweekly applications) were retrieved. 11 were male and 10 female, with ages ranging from 5 to 25 years (median 15.5 years). The mandible was the most frequent localization (13 cases) followed by the maxilla (8 lesions). According to the criteria established by Chuong et al., the lesions from 10 patients were classified as aggressive, and 11 were classified as non-aggressive.

The treatment response was determined using previously determined scores in which four criteria were considered: stabilization or regression of lesion size evaluated clinically and in follow-up radiographs; the absence of symptoms; increased radio-opacity in radiographs, representing peripheral and/or central calcification of the lesion; increased difficulty in solution infiltrating the lesion during the sequence of applications. When a case met all four criteria, the response was determined to be good; meeting two or three criteria was determined to be moderate; and meeting one criterion or no criteria implied a negative response to treatment. On the basis of these criteria, 15 patients had a good response, 4 had a moderate response, and 2 had a negative response. Although these criteria present some subjective topics, such as ‘increased difficulty of the injection’, the treatment is usually performed by the same surgeon so increasing the difficulty of the injection can be measured. This finding is reported by several authors, and it reflects the ossification of the lesion. It is recommended that the surgeon takes notes about the resistance of the injections.

Immunostaining was performed according to a previously described protocol. For antigen retrieval, deparaffinized sections were pretreated by heating in a microwave oven in 10 mM citrate buffer pH 6.0 for 15 min. After cooling, the sections were immersed in phosphate buffered saline (PBS) containing 3% hydrogen peroxide for 10 min to block endogenous peroxidase activity. The sections were then incubated in a humid chamber overnight at 4 °C with the following primary antibodies: anti-glucocorticoid receptor (NCL-GCR; dilution 1:50; Vision-Biosystems ^® , UK) or anti-calcitonin receptor (anti-calcitonin receptor precursor antibody; dilution 1:50; Novus Biologicals ^® , USA). After rinsing with PBS, the slides were incubated with secondary antibody followed by streptavidin–peroxidase complex both for 30 min at room temperature with a PBS wash between each step (LSAB+ system; DakoCytomation ^® , USA). The slides were developed with diaminobenzidine–H ₂ O ₂ (DAB+ system; DakoCytomation ^® , USA), counterstained with Harry’s haematoxylin and mounted. Thyroid tissue for CTR and tonsil tissue for GR were used as positive controls. Controls of primary antibody specificity included the omission or substitution of primary antiserum with normal bovine serum.

The immunohistochemical staining was assessed using a direct light microscope, accordingly to the site of stain of each antibody (GR, nuclei; CTR, nuclei and cytoplasm). A differential count was performed for MSCs and MGCs. Staining was quantified through manual counting of at least 1000 MSCs in 10 different fields at a magnification of 400×. In the same 10 high-power fields, all MGCs were counted to a maximum of 1000 cells. The labelling index (LI) was expressed as the percentage of positive cells with nuclear staining for CTR in each section. The H -score ( H ) takes into account the intensity of the cytoplasmic CTR staining and nuclear GR staining expressed in values ranging from 0 to 3+ (0, no stain; 1+, weak; 2+, moderate; 3+, strong) in accordance with the methods described by McCarty et al.