Autophagy and immune microenvironment in craniopharyngioma and ameloblastoma.

BACKGROUND: Craniopharyngiomas and ameloblastomas show remarkable histologic and molecular similarities. The immune microenvironment of craniopharyngiomas has been recently studied showing interesting findings, while its composition in ameloblastomas is unknown. Similarly, some evidence of autophagic activity, a process of cellular constituents' degradation has been found in ameloblastomas, but no studies exist in craniopharyngiomas. Thus, the aim of the study is to compare factors of the immune microenvironment and the autophagic apparatus between these two tumor types. METHODS: 26 craniopharyngiomas and 14 ameloblastomas were immunohistochemically studied for PD-L1, CD8, CD20, S100, CD163, MECA-79, LC3B and p62. RESULTS: Craniopharyngiomas showed higher LC3B tumor cell expression, higher CD8+ T cells and higher CD163+ macrophages in comparison to ameloblastomas. LC3B tumor cell expression was associated with overall survival in craniopharyngioma patients and p62 nuclear expression was associated with overall survival in ameloblastoma patients. CONCLUSION: This is the first study showing the presence of autophagic markers in craniopharyngiomas and describing the immune microenvironment of ameloblastomas.

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 ß-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 ß-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.

Immunoexpression of BMP-2 and BMP-4 and their receptors, BMPR-IA and BMPR-II, in ameloblastomas and adenomatoid odontogenic tumors.

OBJECTIVES: The present study evaluated the immunohistochemical expression of BMP-2 and BMP-4 and of their receptors (BMPR-IA and BMPR-II) in solid ameloblastoma (SA), unicystic ameloblastoma (UA) and adenomatoid odontogenic tumor (AOT) in order to obtain a better understanding of their role in the development and biological behavior of these tumors. DESIGN: This study analyzed these proteins in 30 cases of SA, 10 cases of UA, and 30 cases of AOT. Immunoexpression was evaluated in the parenchyma and stroma by attributing the following scores: 0, no stained cells; 1, ≤10%; 2, >10% and ≤25%; 3, >25% and ≤50%; 4, >50% and ≤75%.; 5, >75% stained cells. RESULTS: In SAs, positive correlations were observed between the stromal and parenchymal expression of BMP-2 (p<0.001) and between the stromal expression of BMP-2 and BMP-4 (p=0.020), as well as between the stromal expression of BMPR-II and BMP-4 (p=0.001) and the stromal and parenchymal expression of BMPR-II (p<0.001). In UAs, correlations were detected between the stromal and parenchymal expression of BMP-4 (p=0.035) and between the stromal expression of BMP-4 and BMPR-IA (p=0.022). In AOTs, analysis of immunoexpression in the parenchyma revealed positive correlations between all proteins. CONCLUSION: BMPs and their receptors play an important role in the differentiation and development of ameloblastomas and AOTs, but may not explain the different biological behaviors of these lesions. The positive correlation observed in AOTs might be related to the formation of mineralized material in this tumor.

Comparison between Manual and Automated Methods for Ki-67 Immunoexpression Quantification in Ameloblastomas.

Ameloblastoma is a common and unpredictable odontogenic tumor with high relapse rates. Several studies assessing the proliferative capacity of these neoplasms have been published, mainly using the protein Ki-67. Cell counts must be completed to determine the cell proliferation rate. Multiple methods have been developed for this purpose. The most widely used method is the labeling index, which has undergone changes over time to better facilitate cell counting. Here, we compared manual cell counting methods with automated cell counting (ImmunoRatio) to determine the relative effectiveness of these methods. The results suggest that ImmunoRatio, a free software tool, may be highly advantageous and provide results similar to manual cell counting methods when used with the appropriate calibration. However, ImmunoRatio has flaws that may affect the labeling index results. Therefore, this automated cell counting method must be supplemented with manual cell counting methods.

Tissue microarray use for immunohistochemical study of ameloblastoma.

BACKGROUND: Ameloblastoma is a locally aggressive odontogenic tumor with high rates of recurrence. To better understand the molecular basis of ameloblastoma, tissue microarray (TMA) may represent a useful tool. However, despite TMA has been considered a high-throughput technique for different human neoplasms, it remains to be validated in the ameloblastoma context. Therefore, the objective of this study was to validate TMA for immunohistochemical study of ameloblastoma, determining its most appropriate design. METHODS: Forty cases of ameloblastoma were manually distributed in two TMA blocks assembled in triplicate containing 1.0- and 2.0-mm cores (20 cases each). Immunohistochemistry for cytokeratins 14 and 19, and Bcl-2 and Ki-67 was performed, and semiquantitative analysis was performed. Results obtained with TMA sections were compared to their corresponding conventional whole-section slides (CWSS). RESULTS: Kappa statistical test demonstrated that both 1.0- and 2.0-mm cores assessed as duplicate or triplicate significantly correlated with CWSS, with higher levels obtained using Ki67 (k = 0.98, 0.97, 0.88, 0.87) and CK19 (k = 0.62, 0.58, 0.85, 0.85). There was no significant difference between 1.0- and 2.0-mm cores, and between duplicate and triplicate values. 1.0-mm TMA showed a higher index of core loss (33.74% vs. 4.99%). CONCLUSION: Using a manual arrayer, it was demonstrated that 1.0-mm TMA arranged in duplicate is a valid method for ameloblastoma immunohistochemical study with satisfactory levels of agreement between TMA cylinders and CWSS.

IMMUNOEXPRESSION OF TWIST IN DIFFERENT MORPHOLOGICAL VARIANTS OF AMELOBLASTOMAS.

BACKGROUND: Ameloblastoma is the most common and a clinically significant odontogenic tumour. The diagnosis and sub classification of ameloblastoma have been traditionally relied on histological assessment, but it is still a subject of debate. The aim of this study was to evaluate the immuno-expression of Twist in various subtypes of ameloblastomas and their corelation with various histological variants. METHODS: This cross-sectional study was conducted in the Department of Pathology, Post Graduate Medical Institute, Lahore between June to December 2013. Thirty cases of various types of Ameloblastomas were included in this study. Histological sub-classification of the tumours was performed based on WHO classification. Twist expression of these tumours was estimated by immunohistochemistry, performed on paraffin sections. RESULTS: Out of these thirty cases, 22 (73%) were newly diagnosed typical solid/multicystic intraosseous ameloblastoma, 2 (7%) cases belonged to recurrent ameloblastoma, and 3 (10%) each were diagnosed as peripheral/extraosseous ameloblastoma and ameloblastic carcinoma. On histopathological sub-classification of the newly diagnosed solid ameloblastoma, 8 cases were diagnosed as follicular ameloblastoma in which 3 cases (37.5%) were negatively stained, 4 cases (50%) were mild positive and 1 case (12.5%) was moderate positive with Twist immunostaining. Out of 5 cases of plexiform ameloblastoma 3 cases (60%) were mild positive. Out of 4 cases of acanthomatous ameloblastoma 2 (50%) were moderate positive and 2 cases (50%) were strong positive. All granular cell ameloblastoma stained positive. The only case of desmoplastic ameloblastoma was moderately positive. Both the cases of recurrent ameloblastoma were strongly positive. All 3 cases of peripheral ameloblastoma stained negatively. All ameloblastic carcinoma were strongly positive. CONCLUSION: Twist immunohistochemical analysis can be used as an adjuvant to H&E histopathological findings for proper categorization and grading of ameloblastoma especially in the clinically aggressive tumours.

Estudo da expressão imuno-histoquímica das proteínas MMP-9, MMP-13, e TIMP-1 em ameloblastomas e tumores odontogênicos ceratocistos / Study of immunohistochemical expression of MMP -9, MMP -13 protein, and TIMP - 1 in ameloblastomas and odontogenic keratocysts tumors

Os ameloblastomas e tumores odontogênicos ceratocísticos (TOC) representam lesões odontogênicas que, apesar de sua natureza benigna, se destacam por um comportamento biológico distinto, caracterizado pelo crescimento localmente agressivo e episódios recidivantes. A reabsorção dos ossos gnáticos provocada pelo crescimento dessas lesões constitui um fator determinante à expansão das mesmas, sendo mediada tanto por células osteoclásticas como pela ação enzimática de diversas metaloproteinases de matriz (MMPs). A expressão de fatores estimuladores e inibidores da reabsorção óssea vem sendo correlacionada com o desenvolvimento destas lesões, merecendo destaque algumas MMPs como as colagenases e as gelatinases e os inibidores teciduais de metaloproteinases (TIMPs), dentre outros. Baseados na premissa de que fatores estimuladores e inibidores de processos osteolíticos podem ser determinantes para o ritmo de crescimento de lesões odontogênicas intraósseas, o objetivo de estudo foi avaliar a imunoexpressão das proteínas MMP-9, -13 e TIMP-1 no epitélio e mesênquima de espécimes de ameloblastomas e TOC. A análise estatística foi realizada através dos testes de Mann-Whitney e Wilcoxon com nível de significância estabelecido em 5%. Através de uma análise quantitativa das células imunomarcadas, foi observada a expressão imuno-histoquímica das MMP-9, -13 e TIMP-1 em 100% dos casos, tanto no epitélio quanto no mesênquima tumoral. Mais de 76% das células epiteliais (escore 3) dos TOC e ameloblastomas apresentaram imunomarcação para MMP-9 (p=0,382) e MMP-13 (p=0,069), sendo estatisticamente significativa para o TIMP-1 (p=0,003) nos ameloblastomas. No mesênquima, observou-se maior escore de imunomarcação da MMP-13 (p=0,031) nos ameloblastomas em relação aos TOC, enquanto para a MMP-9 e TIMP-1 não se observou diferença estatisticamente significativa (p=0,403; p=1,000). O cálculo da razão entre os escores de expressão das proteínas revelou, de uma maneira geral, similaridade entre as lesões, sendo observado predomínio significante de igualdade de expressão do TIMP-1 e da MMP-9 apenas no epitélio dos ameloblastomas. A imunoexpressão marcante das MMP-9, MMP-13 e TIMP-1 no epitélio e mesênquima das lesões estudadas indica que estas proteínas participam na remodelação da MEC necessária à progressão tumoral, no entanto, as diferenças pontuais observadas na expressão de algumas destas proteínas, não são suficientes para sugerir diferenças no comportamento biológico dos ameloblastomas e dos TOCs. (AU)

Ameloblastomas and keratocystic odontogenic tumors (KOT) represent odontogenic lesions that, despite their benign nature, are distinguished by a distinct biological behavior, characterized by locally aggressive growth and recurrent episodes. The gnathic bone resorption caused by the growth of these lesions is a key to the expansion of the same, both being mediated by osteoclastic cells like enzymatic activity of various matrix metalloproteinases (MMPs) factor. The expression of stimulatory factors and inhibitors of bone resorption has been correlated with the development of these lesions, with emphasis to some MMPs such as collagenases and gelatinases and tissue inhibitors of metalloproteinases (TIMPs), among others. Based on the premise that stimulatory and inhibitory factors of osteolytic processes can be decisive for the growth rate of intraosseous odontogenic lesions, this experiment evaluated the immunoreactivity of MMP-9, -13 and TIMP-1 protein in the epithelium and mesenchyme of ameloblastoma and the KOT specimens, by a quantitative analysis of the immunoreactivity cells. Statistical analysis was performed using the MannWhitney and Wilcoxon tests with a significance level set at 5 %. Immunohistochemical expression of MMP-9, -13 and TIMP-1 was observed in 100% of cases both in the epithelium and in mesenchyme. The immunoreactivity in the epithelium of KOT and ameloblastomas revealed a predominance of score 3 for MMP-9 (p=0.382) and MMP-13 (p=0.069) and no statistically significance for TIMP-1, the latter being significantly higher immunoreactivity in ameloblastomas. In the mesenchyme, there was a higher score immunoreactivity of MMP-13 (p=0.031) in ameloblastomas in relation to KOT, whereas for MMP-9 and TIMP-1 no statistically significant difference (p=0.403 was observed, p=1.000). The calculation of the ratio of scores revealed expression of proteins in general, similarity of the lesions, a significant predominance of equal expression of TIMP-1 and MMP-9 was observed only in the epithelium of ameloblastoma. The marked immunostaining of MMP-9 , MMP-13 and TIMP-1 in epithelium and mesenchyme of the lesion indicate that these proteins involved in ECM remodeling required for tumor progression, however, specific differences in the expression of some of these proteins, are not sufficient to suggest differences in the biological behavior of ameloblastomas and KOTs. (AU)

Expresión inmunohistoquímica de PCNA, Ki-67 y ciclina D1 en ameloblastomas multiquísticos / Immunohistochemical expression of PCNA, Ki-67 and cyclin D1 in solid ameloblastomas

Introducción: el ameloblastoma es un tumor odontogénico benigno, localmente agresivo, que debe su origen a partir de estructuras epiteliales involucradas en la odontogénesis. El objetivo del presente trabajo es identificar, por medio de técnicas inmunohistoquímicas, aspectos de los mecanismos regulatorios de proliferación celular y la relación de los diferentes subtipos histológicos con el comportamiento biológico de estos tumores. Materiales y métodos: se seleccionaron 10 ameloblastomas multiquísticos en los cuales se realizó inmunotinción con los marcadores PCNA, Ki-67 y Ciclina D1. La interpretación de las tinciones se basó en la intensidad, localización y los subtipos celulares. La valoración utilizada para contabilizar el número de células fue baja (menos del 10 por ciento), media (hasta el 50 por ciento) y alta (más del 50 por ciento). Resultados: la tinción fue positiva en 6 casos para PCNA, en 3 para Ki-67 y en 5 para ciclina D1, en las células basales periféricas, en los patrones foliculares y plexiformes, en las del esbozo del retículo estrellado y fue negativa en los patrones quísticos y acantomatosos. Conclusión: en base a los hallazgos se puede asumir que las células basales y parabasales de los patrones foliculares y plexiformes presentan mayor actividad proliferativa que otros patrones y determinarían la evolución y tratamiento

Expresión inmunohistoquímica de PCNA, Ki-67 y ciclina D1 en ameloblastomas multiquísticos / Immunohistochemical expression of PCNA, Ki-67 and cyclin D1 in solid ameloblastomas

Introducción: el ameloblastoma es un tumor odontogénico benigno, localmente agresivo, que debe su origen a partir de estructuras epiteliales involucradas en la odontogénesis. El objetivo del presente trabajo es identificar, por medio de técnicas inmunohistoquímicas, aspectos de los mecanismos regulatorios de proliferación celular y la relación de los diferentes subtipos histológicos con el comportamiento biológico de estos tumores. Materiales y métodos: se seleccionaron 10 ameloblastomas multiquísticos en los cuales se realizó inmunotinción con los marcadores PCNA, Ki-67 y Ciclina D1. La interpretación de las tinciones se basó en la intensidad, localización y los subtipos celulares. La valoración utilizada para contabilizar el número de células fue baja (menos del 10 por ciento), media (hasta el 50 por ciento) y alta (más del 50 por ciento). Resultados: la tinción fue positiva en 6 casos para PCNA, en 3 para Ki-67 y en 5 para ciclina D1, en las células basales periféricas, en los patrones foliculares y plexiformes, en las del esbozo del retículo estrellado y fue negativa en los patrones quísticos y acantomatosos. Conclusión: en base a los hallazgos se puede asumir que las células basales y parabasales de los patrones foliculares y plexiformes presentan mayor actividad proliferativa que otros patrones y determinarían la evolución y tratamiento (AU)

Does cytokine profiling of aspirate from jaw cysts and tumors have a role in diagnosis?

PURPOSE: The objective of the present study was twofold: first, to assess aspirates for use in cytokine profiling and second, to initiate pilot analyses to determine whether the cytokine profiling can serve as an aid in the diagnosis of jaw lesions. MATERIALS AND METHODS: The aspirates from 12 benign odontogenic cysts and tumors of the jaw were collected and randomized, and a formal incisional biopsy was performed to establish the tissue diagnosis. The biopsies revealed keratocystic odontogenic tumor, ameloblastoma, and dentigerous cyst. The cystic aspirate was analyzed using the Q-Plex Human Cytokine Screen to detect cytokine expression and determine the level of expression for each pathologic entity. An array of 16 cytokines was investigated, including interleukin (IL)-1α, IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-15, IL-17, IL-23, interferon-γ, tumor necrosis factor (TNF)-α, and TNF-ß. Tables were developed to determine the ratio of expression for the candidate cytokine pairs that were differentially expressed among the 3 pathologic entities encountered. One-way analysis of variance was used to search for significant differences in the ratio of expression of the candidate pairs among the 3 entities. RESULTS: Cytokines expressed by the 3 distinct jaw lesions were detected in the aspirate without the need for tissue biopsy. Cytokine profiling of these entities is possible owing to differential expression of the various cytokines studied. The ratio of expression was significant (P < .05) for 15 pairs of cytokines: IL-5/IL-1α, IL-4/IL-2, IL-8/IL-4, TNF-ß/IL-6, IL-23/IL-6, TNF-α/IL-23, TNF-α/TNF-ß, TNF-α/IL-8, TNF-ß/IL-5, TNF-ß/TNF-α, TNF-ß/IL-13, IL-12/IL-23, IL-13/IL-15, IL-15/IL-2, and IL-6/IL-2. A comparison of the mean values indicated a "high/low" expression value for each lesion type for the 15 cytokine pairs. CONCLUSIONS: Cytokines, expressed by the 3 groups of jaw lesions, can be detected in the cystic aspirate, and a comparison of the ratio of the expression of the aspirates demonstrated a differential expression pattern of cytokines among the 3 groups. These ratios could assist in establishing a prompt and accurate diagnosis of lesions that might be difficult to discern clinically and radiographically. The use of a simple, minimally invasive aspiration procedure can help to establish an accurate diagnosis.

The immunoprofile of odontogenic keratocyst (keratocystic odontogenic tumor) that includes expression of PTCH, SMO, GLI-1 and bcl-2 is similar to ameloblastoma but different from odontogenic cysts.

BACKGROUND: The aggressive biological behavior of odontogenic keratocysts (OKCs), unlike that of other odontogenic cysts, has argued for its recent re-classification as a neoplasm, 'keratocystic odontogenic tumor'. Identification of mutations in the PTCH gene in some of the OKCs that were expected to produce truncated proteins, resulting in loss of control of the cell cycle, provided additional support for OKCs having a neoplastic nature. METHODS: We investigated the immunohistochemical expression of the sonic hedgehog (SHH) signaling pathway-related proteins, PTCH, smoothened (SMO) and GLI-1, and of the SHH-induced bcl-2 oncoprotein in a series of primary OKC (pOKC), recurrent OKC (rOKC) and nevoid basal cell carcinoma syndrome-associated OKCs (NBCCS-OKCs), and compared them to solid ameloblastomas (SAMs), unicystic ameloblastomas (UAMs), 'orthokeratinized' OKCs (oOKCs), dentigerous cysts (DCs) and radicular cysts (RCs). RESULTS: All studied lesions expressed the SHH pathway-related proteins in a similar pattern. The expression of bcl-2 in OKCs (pOKCs and NBCCS-OKCs) and SAMs was significantly higher than in oOKCs, DCs and RCs (P < 0.001). CONCLUSIONS: The present results of the immunoprofile of OKCs (that includes the expression of the SHH-related proteins and the SHH-induced bcl-2 oncoprotein) further support the notion of OKC having a neoplastic nature. As OKCs vary considerably in their biologic behavior, it is suggested that the quality and quantity of interactions between the SHH and other cell cycle regulatory pathways are likely to work synergistically to define the individual phenotype and corresponding biological behavior of this lesion.

CD10 expression in stromal cells of ameloblastoma variants.

OBJECTIVE: We performed an immunohistochemical study in a series of ameloblastomas with different histology to explore the existence of a correlation between CD10 immunoreactivity in peritumoral stromal cells and the type of ameloblastoma with a high risk of local recurrence. STUDY DESIGN: A total of 45 ameloblastomas (18 unicystic [UA], 4 peripheral [PA], 23 solid/multicystic [SA]) were evaluated. Cases showing immunoreactivity for CD10 in < and > or =10% of stromal cells around tumoral epithelial islands, were considered, respectively, negative and positive. Correlations between stromal CD10 expression and histopathologic types with low and high risk of recurrence were evaluated by statistical analysis. RESULTS: SA cases showed a significantly higher percentage of stromal CD10-positive cells than the UA and PA variants. A strong intensity of immunostaining was observed only in SA. CONCLUSIONS: Our results suggest that CD10 expression might be associated with stromal invasion in ameloblastoma variants with a high risk of recurrences.

Aspecto clinicopatológico e inmunohistoquímico en tres casos de ameloblastoma / Clinico-pathologic and immunohistochemical aspects in three ameloblastoma cases

El ameloblastoma es una de las neoplasias odontogénicas más comunes que se presentan en el maxilar inferior con mayor frecuencia, sector posterior, con localización intraósea y periférica (esta última, más rara). Los subtipos histológicos no influencian directamente en el tratamiento ni en el pronóstico porque generalmente se utilizan actos quirúrgicos radicales por la frecuencia de recidiva de los ameloblastomas. Se utilizó en biopsias las técnicas inmunohistoquímicas básicas y Mib-1 (Ki 67 en parafina) para determinar el índice de proliferación celular que indicaría la posible transformación maligna del ameloblastoma o su capacidad de recidiva. En un germen dentario, en estadio de campana, el órgano del esmalte mostró idéntica afinidad inmunohistoquímica con un índice de proliferación celular Mib 1 (Ki 67) de un 1 por ciento (AU)

Ki-67 expression in dentigerous cysts, unicystic ameloblastomas, and ameloblastomas arising from dental cysts.

This study investigated whether or not an ameloblastoma developing in the wall of a dentigerous cyst is a distinct lesion from the unicystic ameloblastoma. An immunohistochemical evaluation of Ki-67 in dentigerous cysts, unicystic ameloblastomas, and ameloblastomas arising in dentigerous cysts was done. The values of Ki-67 positivity were 3.14 for the dentigerous cyst, between 5.32 and 16.56 for unicystic ameloblastoma, and 11.77 for ameloblastoma arising in a dentigerous cyst. Statistically significant differences were found between the dentigerous cyst and the unicystic ameloblastoma and between the dentigerous cyst and the ameloblastoma arising from a dentigerous cyst. No statistically significant difference was present between unicystic ameloblastoma and ameloblastoma arising from dentigerous cyst. These immunohistochemical data confirm the hypothesis that an ameloblastoma arising from a dentigerous cyst has a similar biological behavior to the unicystic ameloblastoma and should be considered as merely a histologic variant.

Histochemistry of Ki-67 antigen in ameloblastomas.

The proliferative potential of two common histologic variants of ameloblastoma was investigated immunohistochemically using Ki-67 antibody on routinely processed, formalin-fixed, paraffin-embedded sections. Thirty cases of ameloblastomas (15 cases of follicular and 15 cases of plexiform type) were analyzed. Autoclave heating pretreatment was employed at 121 degrees C for 20 min prior to analysis. This retrieval method allowed immunoreactive sites of the Ki-67 antigen to become exposed and thus available for immunohistochemical reaction. We found that expressed Ki-67 antigen was localized within the nucleus of tumor cells in both follicular and plexiform ameloblastomas. Immunoreactive cells were localized in the peripheral area of tumor islands as well as in the central stellate reticulum-area. The labeling rate was higher in the plexiform (3.68%) than in the follicular type (1.78%). Results suggest that cell proliferation of ameloblastoma was different depending on histologic variation of the tumor. Further, the proliferative potential was higher in the plexiform ameloblastoma than that in the follicular type.

In situ hybridisation and immunocytochemical localisation of osteolytic cytokines and adhesion molecules in ameloblastomas.

Ameloblastomas produce interleukin-1-like activity that could explain some part of their osteolytic capability. However, the cellular source of this osteolytic activity is unknown. In the present study, cytokines with known inflammatory and osteolytic activity, i.e., interleukin-1 (IL-1), tumour necrosis factor (TNF), and interleukin-6 (IL-6), have been localised by immunocytochemistry and in situ hybridisation. The cellular adhesion receptors ICAM-1, E-selectin and VCAM-1 have also been immunolocalised. Immunocytochemistry demonstrated that all seven specimens showed positive staining for IL-1alpha and IL-6 with these cytokines being located in the stellate reticulum-like cells and vascular endothelium. Very faint staining for IL-1beta was seen in four of seven specimens. No reaction was seen for TNF-alpha. All specimens demonstrated E-selectin staining in the vascular endothelium and ICAM-1 and VCAM-1 staining in the stellate reticulum-like cells and the endothelium. In situ hybridisation for the cytokines showed the presence of mRNA of both IL-1alpha and IL-6 in the stellate reticulum-like cells. Faint staining for IL-1beta was also seen. No staining was seen for TNF. These findings show that ameloblastomas synthesize two bone-modulating cytokines, IL-1alpha and IL-6, and that these are synthesized mainly by the stellate reticulum-like cells. These tumours also contain a proportion of activated blood vessels in which endothelial cells express the cellular adhesion receptors ICAM-1, E-selectin and VCAM-1.

Comparison of proliferating cell nuclear antigen expression in odontogenic keratocyst and ameloblastoma: an immunohistochemical study.

Proliferating cell nuclear antigen (PCNA) is a nuclear protein synthesized in the late G1 and S phase of the cell cycle, and immunohistochemical detection of the protein represents a useful marker for the proliferating fraction of cells in tissue specimens. PCNA expression was studied in odontogenic keratocysts (n = 15) and ameloblastomas (n = 46) using an avidin-biotin-peroxidase complex method on routinely processed paraffin sections. The percentage of PCNA-positive cells determined by point counting was significantly lower in the ameloblastomas (mean 9.4%, standard deviation (SD) 11.0) than in odontogenic keratocysts (mean 29.9%, SD 24.0). In ameloblastomas, the mean percentage of PCNA-positive cells was lowest in the acanthomatous pattern and highest in plexiform pattern. The mean percentage of PCNA-positive cells in plexiform pattern was non-significantly higher than that in follicular pattern. The mean percentage of PCNA-positive cells in plexiform and follicular patterns was significantly higher than that in cystic and acanthomatous patterns. The frequency of PCNA-positive cells was significantly higher in the peripheral cells of follicular and plexiform patterns than in the central cells of both patterns (p < 0.01). Therefore, peripheral cells were regarded as reserve cell of central cells. The mean percentage of PCNA-positive cells in the epithelial lining of odontogenic keratocyst was not significantly different from those in the peripheral cells of follicular and plexiform patterns of ameloblastoma. In contrast, the odontogenic keratocyst exhibited a mean percentage of PCNA-positive cells which was statistically higher than that in other histological elements of ameloblastomas. The present study suggests that odontogenic keratocyst is regarded as benign odontogenic tumour.

Characterization of novel monoclonal antibodies raised against formalin-fixed, paraffin-embedded human ameloblastoma.

To obtain monoclonal antibodies reactive with odontogenic but not other types of epithelium, mice were immunized with homogenates of fixed ameloblastoma tissues, and monoclonal antibodies Y4 and M11 were produced. Y4 reacted immunohistochemically with odontogenic epithelial components but not with those of squamous differentiation, while M11 reacted with odontogenic epithelial components and a part of keratotic epithelial tissues. Immunoglobulin isotypes of both antibodies were IgM as determined by Ouchterlony immunodiffusion and enzyme-linked immunosorbent assays. Western blotting revealed that the antigen recognized by Y4 had a molecular mass of approximately 66 kDa; however, the antigen reactive with M11 was not identified by Western blotting in spite of various attempts in changing reaction conditions. These antibodies may be beneficial to histological analyses of odontogenic tissues and their related lesions.