Joseph A. Regezi

Odontogenic cysts, odontogenic tumors, fibroosseous, and giant cell lesions of the jaws.

Odontogenic cysts that can be problematic because of recurrence and/or aggressive growth include odontogenic keratocyst (OKC), calcifying odontogenic cyst, and the recently described glandular odontogenic cyst. The OKC has significant growth capacity and recurrence potential and is occasionally indicative of the nevoid basal cell carcinoma syndrome. There is also an orthokeratinized variant, the orthokeratinized odontogenic cyst, which is less aggressive and is not syndrome associated. Ghost cell keratinization, which typifies the calcifying odontogenic cyst, can be seen in solid lesions that have now been designated odontogenic ghost cell tumor. The glandular odontogenic cyst contains mucous cells and ductlike structures that may mimic central mucoepidermoid carcinoma. Several odontogenic tumors may provide diagnostic challenges, particularly the cystic ameloblastoma. Identification of this frequently underdiagnosed cystic tumor often comes after one or more recurrences and a destructive course. Other difficult lesions include malignant ameloblastomas, calcifying epithelial odontogenic tumor, squamous odontogenic tumor, and clear-cell odontogenic tumor. Histologic identification of myxofibrous lesions of the jaws (odontogenic myxoma, odontogenic fibroma, desmoplastic fibroma) is necessary to avoid the diagnostic pitfall of overdiagnosis of similar-appearing follicular sacs and dental pulps. Fibroosseous lesions of the jaws show considerable microscopic overlap and include fibrous dysplasia, ossifying fibroma, periapical cementoosseous dysplasia, and low-grade chronic osteomyelitis. The term fibrous dysplasia is probably overused in general practice and should be reserved for the rare lesion that presents as a large, expansile, diffuse opacity of children and young adults. The need to use clinicopathologic correlation in assessing these lesions is of particular importance. Central giant cell granuloma is a relatively common jaw lesion of young adults that has an unpredictable behavior. Microscopic diagnosis is relatively straightforward; however, this lesion continues to be somewhat controversial because of its disputed classification (reactive versus neoplastic) and because of its management (surgical versus. medical). Its relationship to giant cell tumor of long bone remains undetermined.

Effect of Duration of Fixation on Quantitative Reverse Transcription Polymerase Chain Reaction Analyses

Increasingly, there is the need to analyze gene expression in tumor tissues and correlate these findings with clinical outcome. Because there are few tissue banks containing enough frozen material suitable for large-scale genetic analyses, methods to isolate and quantify messenger RNA (mRNA) from formalin-fixed, paraffin-embedded tissue sections are needed. Recovery of RNA from routinely processed biopsies and quantification by the polymerase chain reaction (PCR) has been reported; however, the effects of formalin fixation have not been well studied. We used a proteinase K-salt precipitation RNA isolation protocol followed by TaqMan quantitative PCR to compare the effect of formalin fixation for 24, 48, and 72 hours and for 1 week in normal (2), oral epithelial dysplasia (3), and oral squamous cell carcinoma (4) specimens yielding 9 fresh and 36 formalin-fixed samples. We also compared mRNA and protein expression levels using immunohistochemistry for epidermal growth factor receptor (EGFR), matrix metalloproteinase (MMP)-1, p21, and vascular endothelial growth factor (VEGF) in 15 randomly selected and routinely processed oral carcinomas. We were able to extract RNA suitable for quantitative reverse transcription (RT) from all fresh (9/9) and formalin-fixed (36/36) specimens fixed for differing lengths of time and from all (15/15) randomly selected oral squamous cell carcinoma. We found that prolonged formalin fixation (>48 h) had a detrimental effect on quantitative RT polymerase chain reaction results that was most marked for MMP-1 and VEGF but less evident for p21 and EGFR. Comparisons of quantitative RT polymerase chain reaction and immunohistochemistry showed that for all markers, except p21, there was good correlation between mRNA and protein levels. p21 mRNA was overexpressed in only one case, but protein levels were elevated in all but one tumor, consistent with the established translational regulation of p21. These results show that RNA can be reliably isolated from formalin-fixed, paraffin-embedded tissue sections and can produce reliable quantitative RT-PCR data. However, results for some markers are adversely affected by prolonged formalin fixation times.

Laser management of oral leukoplakias: A follow-up study of 70 patients

University of California, Department of Stomatology, School of Dentistry, San Francisco, California.

Cell cycle proteins and the development of oral squamous cell carcinoma

Expression of cell cycle regulatory proteins was evaluated in premalignant and malignant oral epithelial lesions, to test the hypothesis that protein regulation of the cell cycle may be altered in the development of oral squamous cell carcinoma. Archived paraffin-embedded specimens (n = 90) from 25 patients with recurrent or persistent lesions were evaluated in immunohistochemically stained sections for cell cycle regulatory proteins p53, Rb, Cyclin D1, p27, and p21. The cell cycle was also evaluated by expression of nuclear protein Ki 67. Sections were graded semiquantitatively using a 0-3 + scale to indicate the percentage of positively stained cells. The initial histologic diagnosis for 17/25 patients was either focal keratosis, mild dysplasia, or moderate dysplasia; the initial diagnosis for the remaining eight patients ranged from severe dysplasia to moderately differentiated squamous cell carcinoma. Thirty-three of 90 specimens showed positive p53 expression, 11 of which were dysplasias. Eighty-nine of 90 specimens, from all stages of disease, showed positive Rb expression. Twenty-three of 90 specimens showed positive Cyclin D1 expression, typically in the later stages (carcinoma) of a patients disease. Eighty-four of 90 specimens showed positive p21 expression; while 55 of 90 specimens were positive for p27. In control mucosa, p27 was highly expressed, while Rb and p21 proteins were expressed at relatively low levels; p53 and Cyclin D1 proteins were largely absent. Generally, staining of p53, Rb, p21, and Ki 67 increased with time in serial biopsies, while p27 showed decreased staining with disease progression. These data show that cell cycle regulatory proteins are altered in both premalignant and malignant disease, and that protein phenotypes are heterogeneous. P53 expression is seen early, and Cyclin D1 expression is seen late in the development of oral premalignant and malignant disease. Expression of p53, Rb, p21 and Ki67 increased, while p27 decreased, with disease progression.

Benign neural tumors of the oral cavity: A comparative immunohistochemical study

To determine if immunohistochemistry can be used as adjunct to the diagnosis and classification of oral benign neural tumors, we stained 77 neurally differentiated tumors with a panel of neural-associated antibodies (S-100 protein, CD57, epithelial membrane antigen, factor XIIIa, CD34, CD68, collagen IV). Using standard histologic criteria, we identified 13 schwannomas, 16 neurofibromas, 23 traumatic neuromas, 16 palisaded and encapsulated neuromas, and 9 granular cell tumors from archived oral pathology specimens. Silver stains showed that neurofibromas, traumatic neuromas, and palisaded and encapsulated neuromas consistently contained axon filaments. Although all neural tumors contained S-100-positive cells, schwannomas and palisaded and encapsulated neuromas contained the most. All tumors expressed CD57; traumatic neuromas were stained intensely and the others stained weakly. The consistent epithelial membrane antigen capsular staining of schwannomas and the absence of factor XIIIa-positive dendritic/spindle cells helped distinguish these tumors from others. Many CD34-positive cells were found in schwannomas, and few were found in palisaded and encapsulated neuromas. Variable numbers CD68-positive cells were seen in all neural tumor types; some of these cells appeared to be macrophages and mast cells, but many were thought to be Schwann cells expressing this antigen. Collagen IV staining, apparently representing basement membrane, was generally a feature of all benign neural tumors. The immunophenotype of the granular cells of the GCTs was S-100+, CD57+, and collagen IV+ supporting the putative neural origin of these tumors. We conclude that neural origin/differentiation of a connective tissue tumor can be confirmed with stains for S-100 protein, epithelial membrane antigen, CD57, and collagen IV. Staining patterns and intensities associated with the panel of antibodies tested can be useful in tumor classification.

Expression of integrin β6 enhances invasive behavior in oral squamous cell carcinoma

Abstract Oral squamous cell carcinoma (SCC) is characterized by invasive growth and the propensity for distant metastasis. The expression of specific adhesion receptors promotes defined interactions with the specific components found within the extracellular matrix (ECM). We previously showed that the αvβ6 fibronectin receptor is highly expressed in oral SCC. Here we forced expression of the β6 subunit into poorly invasive SCC9 cells to establish the SCC9β6 cell line and compared these two cell lines in several independent assays. Whereas adhesion to fibronectin was unaffected by the expression of β6, migration on fibronectin and invasion through a reconstituted basement membrane (RBM) were both increased. Function-blocking antibodies to αvβ6 (10D5) reduced both migration on fibronectin and invasion through an RBM, whereas anti-α5 antibodies were effective only in suppressing migration on fibronectin, not invasion. Expression of β6 also promoted tumor growth and invasion in vivo and modulated fibronectin matrix deposition. When grown as a co-culture with SCC9 cells, peritumor fibroblasts (PTF) organized a dense fibronectin matrix. However, fibronectin matrix assembly was decreased in co-cultures of SCC9β6 cells and PTF and this decrease was reversed by the addition of function-blocking anti-αvβ6 antibodies. The expression of β6 also resulted in increased levels of matrix metalloproteinase 3. Addition of the general MMP inhibitor GM6001 to SCC9β6/PTF co-cultures dramatically increased fibronectin matrix assembly in a similar fashion as incubation with anti-αvβ6 antibodies. These results demonstrate that expression of β6 (1) increases oral SCC cell motility and growth in vitro and in vivo; (2) negatively affects fibronectin matrix assembly; and (3) stimulates the expression and activation of MMP3. We suggest that the integrin αvβ6 is a key component of oral SCC invasion and metastasis through modulation of MMP-3 activity.

Oral submucosal dendrocytes: factor XIIIa+ and CD34+ dendritic cell populations in normal tissue and fibrovascular lesions

Factor XIIIa+ and CD34+ dendritic cells, believed to be subsets of monocyte/macrophages, have been identified in dermis and in dermal tumors. The purpose of this study was to determine the presence and distribution of analogous cell types in oral sub‐mucosa and oral fibro‐vascular lesions. Antibodies to XIIIa, CD34, S‐100 protein, and macrophage antigen (MAC 387) were tested on formalin‐fixed, paraffin‐embedded tissue sections from normal mucosa, peripheral fibroma (PF), peripheral ossifying fibroma (POP), peripheral giant cell granuloma (PGCG), pyogenic granuloma (PG), lymphangioma (La), benign fibrous histiocytoma (BFH), idiopathic histiocytosis (IH), angiofibroma (Af) using an ABG immunoperoxidase technique. Numbers of positively stained cells were compared to unstained cells in the tumors. XII la positive submucosal dendrocytes (GD34‐, S‐100‐, MAC 387‐) were found in abundance in normal tissue in characteristic distributions: collagen‐associated, vessel‐associated, and lymphoid‐associated. The percentage of XIIIa+ cells in the oral tumors was as follows: PF: 10–30%, POF: 5–10%, PGCG: 0–5%, PG: 5–20%, La: 0%, BPH: 5–25%, IH: 0%, and Af: 10–20%. CD34+ dendrocytes (XIIIa‐, S‐100‐, MAC 387‐) were few in number and were found in deeper submucosa, especially around skeletal muscle. Other than blood vascular endothehum, CD34+ cells were not generally seen in the oral tumors studied. It is concluded that two previously unrecognized dendrocyte populations reside in normal submucosa. XIIIa+ cells participate in the formation of some oral reactive and neoplastic lesions.

p53 gene mutations in sequential oral epithelial dysplasias and squamous cell carcinomas

Previous studies of oral cancer have suggested that alterations of the p53 tumour suppressor gene occur early in the precancerous stage of development. However, these observations have been based on cross‐sectional assessment of abnormal p53 protein staining by immunohistochemistry and may not necessarily reflect gene changes. The purpose of this longitudinal study was to examine the changes in the p53 gene in progressive, sequential epithelial dysplasias and carcinomas from the oral cavity. The study analysed 24 formalin‐fixed, paraffin‐embedded tissue biopsies from ten patients with two or more temporally distinct lesions from the same site in the oral cavity with the diagnosis of hyperkeratosis, epithelial dysplasia, carcinoma in situ or squamous cell carcinoma. Exons 5–8 of the p53 gene were amplified from genomic DNA using intronic primers and directly sequenced using fluorescent‐labelled primers. Standard immunohistochemistry with the DO7 monoclonal antibody was used to detect mutant and wild‐type p53 protein. Mutations of the p53 gene were identified in 9 of 24 samples. Eight were missense mutations and one occurred at a splice site. In six patients, mutations of the p53 gene occurred late after the transformation of epithelial dysplasia to carcinoma. In two patients with progressive dysplasia, but who had yet to develop invasive carcinoma, p53 missense mutations occurred at the carcinoma in situ stage in one case and in a moderate dysplasia in the other. There was an inconsistent relationship between gene mutations and the level of p53 protein staining by immunohistochemistry. It is concluded that during oral carcinogenesis, p53 gene mutations seem to occur relatively late and are associated with transformation to the invasive phenotype. Copyright

Tenascin and β6 integrin are overexpressed in floor of mouth in situ carcinomas and invasive squamous cell carcinomas

Floor of the mouth squamous cell carcinomas exhibit many characteristics that suggest they represent a distinct biological subset within head and neck tumors. The features of preinvasive lateral intraepithelial spread, high rate of conversion of intraepithelial neoplasia to invasive carcinoma, and high incidence of occult metastases, suggest the importance of motility-associated proteins in the pathogenesis of these lesions. Two such proteins, tenascin and beta 6 integrin, are generally overexpressed in squamous carcinomas, and may play a central role in the invasive process of floor of the mouth lesions. The purpose of this study was to evaluate in situ and invasive squamous cell carcinomas from the floor of the mouth for the expression of tenascin and beta 6 integrin. Twenty lesions each of floor of the mouth in situ carcinomas and squamous cell carcinomas, and 10 normal controls were stained for tenascin and beta 6 using a standard immunohistochemical protocol for formalin-fixed specimens. Sections were assessed for staining intensity, pattern, and co-localization. Tenascin was highly expressed at the keratinocyte-connective tissue interface of both in situ and invasive carcinomas. beta 6 was expressed in basal keratinocytes of all in situ and invasive lesions, but was not evident in any of the control epithelia. There was no significant difference in staining of in situ and invasive carcinomas, but there was a significant difference in staining between these lesions and controls. Staining was colocalized in serial sections, supporting a receptor-ligand relationship. Both tenascin and beta 6 were weakly expressed in dysplastic areas adjacent to carcinomas suggesting that changes in the expression of these proteins occurs prior to the invasive phenotype. We conclude that tenascin and beta 6 are overexpressed in in situ and invasive floor of the mouth carcinomas, but that transgression of the basement membrane by neoplastic epithelial cells requires additional changes to the keratinocyte molecular profile.

Salivary Gland Basal Cell and Canalicular Adenomas Immunohistochemical Demonstration of Myoepithelial Cell Participation and Morphogenetic Considerations

OBJECTIVE To evaluate cellular composition of salivary gland adenomas using 3 monoclonal antibodies that recognize a smooth muscle phenotype confirmed to be sensitive for myoepithelial differentiation. DESIGN Immunohistochemical evaluation of 25 salivary gland basal cell and canalicular adenomas. SETTING Archival pathology material from the files of Henry Ford Hospital, Detroit, Mich, and the University of California at San Francisco. RESULTS All basal cell adenoma variants exhibit some degree of myoepithelial cell participation with periductal, epithelioid, and spindled (stromal-like) morphologic structures. Only the canalicular adenomas, even if mixed with trabecular and solid patterns, are devoid of staining with these 3 antibodies, suggesting an adenoma composed exclusively of ductal luminal cells. CONCLUSIONS There is an overlapping histomorphologic and common cellular composition of the basal cell adenoma variants with other recognized adenomas, such as pleomorphic adenoma and myoepithelioma. Relative differentiation toward 3 cell phenotypes (ductal luminal, basal, and myoepithelial) and the character of extracellular matrix production in varying proportions by the neoplastic myoepithelial cells distinguishes the spectrum of salivary gland adenomas identified in current classification schemes.