Comparative histological and immunohistochemical study of ameloblastomas and ameloblastic carcinomas

Background This study aimed to compare the histological and immunohistochemical characteristics of ameloblastomas (AM) and ameloblastic carcinomas (AC). Material and Methods Fifteen cases of AM and 9 AC were submitted to hematoxilin and eosin (H&E) and immunohistochemical analysis with the following antibodies: cytokeratins 5,7,8,14 and 19, Ki-67, p53, p63 and the cellular adhesion molecules CD138 (Syndecan-1), E-cadherin and β-catenin. The mean score of the expression of Ki-67 and p53 labelling index (LIs) were compared between the groups using the t test. A value of p<0.05 was considered to be statistically significant. Results All cases were positive for CKs 5, 14 and 19, but negative for CKs 7 and 8. CKs 5 and 19 were positive mainly in the central regions of the ameloblastic islands, while the expression in AC was variable in intensity and localization. CK14 was also variably expressed in both AM and AC. Ki-67 (P=.001) and p53 (P=.004) immunoexpression was higher in AC. All cases were positive for p63, but values were higher in AC. CD138 was mainly expressed in peripheral cells of AM, with a weak positivity in the central areas, while it was positive in most areas of ACs, except in less differentiated regions, where expression was decreased or lost. E-cadherin and β-catenin were weakly positive in both AM and AC. Conclusions These results shows that Ki-67, p53 and p63 expression was higher in AC as compared to AM, suggesting that these markers can be useful when considering diagnosis of malignancy, and perhaps could play a role in malignant transformation of AM. Pattern of expression of CKs 5 and 19 in AC were different to those found in AM, suggesting genetic alterations of these proteins in malignant cells. It was confirmed that CK19 is a good marker for benign odontogenic tumors, such as AM, but it is variably expressed in malignant cases. Key words:Ameloblastoma, ameloblastic carcinoma, immunohistochemistry, odontogenic tumors.


Introduction
Ameloblastoma (AM) is one of the most common benign odontogenic neoplasms of the jaws, affecting mainly the posterior region of the mandible. It is locally aggressive, with a high rate of recurrence, while ameloblastic carcinoma (AC) is considered its malignant counterpart. Although rare, with less than 120 cases reported in the English literature, AC is the most common malignant odontogenic tumor (1). Microscopically it may show areas reminiscent of ameloblastoma, but with an invasive and less organized pattern, presenting atypia, mitoses and loss of typical ameloblastic morphology (2). Diagnosis of AC sometimes can be difficult, and both, clinical and histopathological features should be considered. The aim of this study was to compare the microscopic and immunohistochemical characteristics of 9 cases of AC and 15 of AM to determine the pattern of expression or differences in location or intensity of expression that may be of assistance in their differentiation.

Material and Methods
Fifteen cases of solid/multicystic AM and 9 AC were retrieved from the files of 5 Latin American oral pathology diagnostic services (Piracicaba Dental School, Brazil; Head and Neck Clinical Center, Guatemala; Peribact Private Service of Oral Pathology, Mexico; Oral Pathology Laboratory of the Universidad Autonoma Metropolitana Xochimilco, Mexico, and Oral Pathol-Conclusions: These results shows that Ki-67, p53 and p63 expression was higher in AC as compared to AM, suggesting that these markers can be useful when considering diagnosis of malignancy, and perhaps could play a role in malignant transformation of AM. Pattern of expression of CKs 5 and 19 in AC were different to those found in AM, suggesting genetic alterations of these proteins in malignant cells. It was confirmed that CK19 is a good marker for benign odontogenic tumors, such as AM, but it is variably expressed in malignant cases.

Results
Among the 15 cases of AM there were 9 males and 6 females; age ranged from 11-58 yr. (mean age: 33.60 yr.). Nine cases involved the posterior mandible, and histologically all were of the solid type, 12 with predominant plexiform and 3 with follicular patterns. The stroma was variably collagenized, and in 4 cases it was focally myxoid. Ki-67 was mainly positive in some cells of the basal and suprabasal layers, but only occasional cells in the central areas similar to the stellate reticulum of the enamel organ were marked. The proliferative index varied between 0.35% and 6.89% with a mean of 2.37% (Fig. 1a). The localization and percentage of p53 immunostaining were similar to Ki-67, but it was less intense, with values of positive nuclei between 0.13% and 5.90% and a mean of 1.77% (Fig. 1b). p63 was positive mainly in the columnar cells of the peripheral layer of the islands/ cords while in the central areas staining was variable, except for those areas of squamous metaplasia, which were positive in most cases (Fig. 1c). Regarding CKs expression, CK5 appeared more intensely expressed in the central stellate reticulum-like areas than in the basal columnar cells, and it was positive in islands presenting flattened cells in both the peripheral and central areas (Fig. 1d), while in areas of squamous metaplasia it was variably expressed. CK14 expression was weak and diffuse in most cases, and e327 positiveness was mainly found in the stellate reticulumlike areas (Fig. 1e), but also in the basal layer of cystic areas, and it was weakly positive in some of the most superficial cells. CK19 showed strong expression in the central cells of the cords/islands, but it was weak or negative in the peripheral columnar cells (Fig. 1f). All cases of AM were negative for CK7 and CK8. Among the adhesion molecules evaluated, CD138 was the most strongly expressed, particularly in the basal and suprabasal layers, while areas of squamous metaplasia were negative or weakly positive, while most central stellate reticulum-like cells were negative (Fig.  1g). E-cadherin expression in AM was not as evident as CD138, and in some cases staining was faint or practically negative. The basal and suprabasal cells exhibited a higher positivity than those located in the central areas. In some cases, groups of cells presenting squamous metaplasia were strongly positive, particularly when cell junctions were evident in H&E (Fig. 1h). β-catenin was the least expressed of the three adhesion markers used, being weakly positive in most cases, except in areas with squamous metaplasia, where expression was more evident (Fig. 1i).
With respect to the 9 cases of AC, there were 5 males and 4 females, with a mean age of 43.67 years (range: 22-65 years). The mandible was involved in 8 cases, 7 in the posterior region, and only one case occurred in the maxilla (Fig. 2). All cases were solid, with only one showing cystic areas. Most cases were composed by sheets of cells arranged in a plexiform pattern, but in 2 cases a follicular pattern predominated. The malignant epithelial cells showed pleomorphism and mitoses, and in six cases there were evidence of focal areas of necrosis. Some islands showed central areas similar to the stellate reticulum of the enamel organ, and most cases contained areas reminiscent of benign ameloblastoma but these were exceeded by the malignant component. The stroma usually was scarce and collagenized (Fig.  3).
Ki-67 proliferative index in AC ranged from 11.87% to 53.29% (mean: 23.46%), with positiveness in both peripheral and central cells (Fig. 4a). Regions similar to benign ameloblastoma exhibited a lower expression of Ki-67, but these were not included for quantitation. The distribution of cells positive for p53 was similar as above described for Ki-67, but the nuclei were much less intensely stained, with percentages of positive cells varying from 11.57% to 80.88% (mean: 36.01%) (Fig.  4b). p63 expression was strongly positive in most cases and in most cells (Fig. 4c). Expression of CK5 in AC was variable, as in 7 cases it was strongly positive either at the periphery or within  the central epithelial cells, while two cases showed total or partial loss of CK5 in most areas, being positive only in a few more differentiated islands still showing ameloblastic characteristics (Fig. 4d). Expression of CK14 was also variable in intensity and localization, with 5 cases presenting positive expression in most of the cells, while in 4 it was either negative or focally positive (Fig.  4e). CK19 expression was not uniform, showing areas strongly positive mainly in a few islands with ameloblastic characteristics (Fig. 4f). Two cases of AC were focally positive for CK7 and CK8. CD138 was strongly positive in most cases, except in very undifferentiated areas where its expression was lost or it was weakly positive (Fig. 4g). Expression of Ecadherin was variable both in intensity and localization (Fig. 4h). β-catenin expression was weak or negative in all but one case (Fig. 4i). Main immunoprofile and comparison between ameloblastomas and ameloblastic carcinoma are shown in table 2.

Discussion
AMs are locally aggressive benign tumors with a relatively high risk of recurrence and potential to malignant transformation into AC. Up to date, there are around 120 cases of AC reported in the English literature, some of which seem to have developed from AM (4-5). Radiographically, AM may appear either as uni or multilocular osteolytic lesions that frequently produce cortical expansion. If paresthesia, pain, irregular borders and invasion of adjacent structures are present, then a malignant lesion should be suspected (2,6). In contrast to AM, ACs usually present microscopic evidence of malignancy, but confirmation of the diagnosis of a secondary type of AC (those developed from a pre-existent AM) need to be supported by a history of persistent or recurrent AM, or the presence of benign residual areas of AM; eventually, differential diagnosis between these two lesions can be difficult, particularly in incisional biopsies, and therefore other types of intraosseous carcinomas of the jaws should be considered. Immunohistochemistry is not usually employed to assist diagnosis and classification of odontogenic tumors, but some immunomarkers may be useful for a better understanding of their origin and possible biological behavior, and therefore the immunohistochemical profile of these lesions may sometimes be helpful to reach  (14) found that both benign and malignant areas of one case of AC were negative for p53 by immunohistochemistry. In our opinion the information obtained in a single case is not enough to compare with our findings, and with those found by the other authors that based their studies in series of cases that have followed a similar sample processing and analysis, with less probabilities of misdiagnosis and technical errors. It is well known that CKs 14 and 19 are regularly expressed in the epithelium of the dental germ, where CK14 is found in the diverse components during all stages of tooth development, including the dental lamina, the inner enamel epithelium and in almost all cells of the enamel organ, while CK19 is mainly positive in the later stages of development, during terminal differentiation of ameloblasts (15,16). We found a similar pattern of expression of these proteins in AM, but it was different to the one observed in AC since in the malignant tumor the expression was irregular in intensity and location for both CKs. CKs 5, which was less expressed in AC than in AM and CK14 seem to be the major CKs High expression in basal and suprabasal cells, but the nuclei were much less intensely stained.
(36.01%) p63 Positive mainly in the columnar cells of the peripheral layer while in the central areas was variable. Squamous metaplasias were positive in most cases.
Strong positiveness in most cases and in most of the cells.

CK5
Strong expression in the central areas of the epithelial islands and weak or negative in the peripheral columnar cells except in the cells with flattened and cuboidal aspect.
Strongly positive at the periphery or within the central epithelial cells, while two cases showed total or partial loss in most areas.

CK14
Weak and diffuse expression in the central areas, while weak or negative in focal areas with squamous metaplasia.
Variable in intensity and localization, with 5 cases presenting positive expression in most of the cells, while in 4 it was either negative or focally positive.

CK19
Strong expression in the central areas and weak or negative in the peripheral columnar cells except in the cells with flattened and cuboidal aspect.
It was not uniform, showing areas strongly positive, mainly in a few islands with ameloblastic characteristics

CD138
Higher expression in the basal and suprabasal layers Strongly positive in most cases, except in very undifferentiated areas where its expression was lost or it was weakly positive E-cad Higher positivity in basal and suprabasal cells than those located in the central areas. In squamous metaplasia focal cells were strongly positive.
Variable either in intensity or localization, with some cases being strongly positive in focal areas while others were considered negative.

-cat
Weakly positive in most cases, except in areas with squamous metaplasia, where expression was more evident.
Weak or negative in all cases. expressed by epithelium in the human enamel organ, marking the end of secretion of amelogenin (17). CK8 is found in embrionary and several simple epithelia, as well as in the human enamel organ, but Crivellini et al., concluded that CK8 was absent in odontogenic epithelium, as it was observed in most cases in our study. CK19 expression has been demonstrated in most odontogenic cysts and benign tumors, and it has been considered a good marker for benign odontogenic lesions (14). Nevertheless it is also expressed in AC and therefore has no diagnostic utility to differentiate among these entities (18,19). In the present study, CK19 expression in AC was variable, being usually maintained in areas with ameloblastic characteristics. p63 is expressed in normal epithelium and it is often overexpressed in carcinomas, including oral squamous cell carcinoma (20). It is also overexpressed in odontogenic carcinomas, such as AC, primary intraosseous squamous cell carcinoma and clear cell carcinoma as compared to benign odontogenic lesions, suggesting that p63 may have a role in tumorigenesis of odontogenic lesions (21). In the present study, p63 was mainly expressed in the basal and suprabasal cells of AM, in a pattern previously observed in odontogenic keratocyst, suggesting that this protein is a good marker of more immature keratinocytes (22). Different from AM, p63 was overexpressed in all our cases of AC, labeling most of the cells, in a similar way as it occur in other types of carcinomas, indicating lack of cell differentiation and a higher potential for growth, two common characteristics of malignancies (23). Cell adhesion molecules and their interactions can be altered in tumors, particularly in malignancies, favoring progression, invasion, recurrence and metastasis (5). In normal conditions, E-cadherin is moderately expressed in odontogenic epithelium during the bell stage, especially in the stellate reticulum of the enamel organ, and also in the polyhedral cells of ameloblastomas (24). β-catenin is a multifunctional molecule that plays a role in cell-cell adhesion mediated by classic cadherins as well as in Wnt-signaling (25). CD138 is a cell surface proteoglycan present in normal epithelial cells, whose expression seems to decrease in ameloblastomas, as compared to epithelial cells of normal enamel organ, having also an inverse relation with cell proliferation index, aggressiveness and recurrence (26-28). There are some studies that have demonstrated positive expression of CD138 in AM, both in the columnar and the polyhedral cells of the stellate reticulum (29), and nu-clear expression of E-cadherin and β-catenin have been reported in cases of solid AM and odontogenic carcinomas (25). In this study, CD138 was the most strongly expressed adhesion molecule, both in AM and AC, while E-cadherin and β-catenin expression was faint in most of the cases. These last two markers were intensely positive only in areas of squamous metaplasia of AMs, particularly when desmosomes were evident in H&E stained preparations. CD138 expression was faint or negative only in the central areas of AMs islands where cells were loosely adhered, leaving spaces between them. CD138 was positive in most areas of AC, except in the less differentiated regions, where expression was decreased or lost. However, our data does not allow us to confirm that there is a higher loss of these molecules in AC as compared to AM, and therefore more studies are necessary to determine if loss of expression of these proteins can be relevant to support malignant transformation or higher aggressiveness in these neoplasms. As mentioned above, expressions of E-cadherin and β-catenin were weak in AMs and ACs, difficulting analysis about their possible role in these neoplasms. In summary, the results of this work confirmed that patients with AC were about a decade older than those with AM. Ki-67 and p53 immunoexpression was higher in AC than in AM, supporting the idea that they can be good markers of malignant transformation. p63 was also more frequently expressed in AC, suggesting loss of maturation of the malignant epithelial cells. The pattern of expression of CKs 5 and 19 is altered in AC in relation to AM, but this does not seem to be related to malignant transformation. Finally, we could not find evident loss of the adhesion molecules CD138, E-cadherin and β-catenin in AC in relation to AM, but as ACs are very rare lesions, we suggest that new series of cases need to be studied for a better understanding of its clinical and biological aspects.