Beta-6 Integrin, tenascin-C, and MMP-1 expression in salivary gland neoplasms.

Beta-6 Integrin, tenascin-C, and MMP-1 (matrix metalloproteinase-1) are invasion-related proteins that are frequently overexpressed in many human malignancies. The objective of this study was to determine whether there is overexpression of these molecules in three types of salivary neoplasms showing markedly different behavior. A total of 55 formalin-fixed, paraffin-embedded archived specimens comprising 19 adenoid cystic carcinomas (ACC), 18 polymorphous low-grade adenocarcinomas (PLGA) and 18 pleomorphic adenomas (PA) were utilized in this study. A standard immunohistochemical technique was used to determine the expression levels of beta-6 integrin, tenascin-C, and matrix metalloproteinase-1 (MMP-1) proteins. Sections were assessed semiquantitatively, and tumors were divided into two groups, low-expressors (0-1+) and high-expressors (2-3+) for statistical analysis. Staining was graded as 0 (<1% positive tumor cells), 1+ (<25% positive tumor cells), 2+ (25-50% positive tumor cells), and 3+ (>50% positive cells). The results showed that the malignant tumors were higher expressors of beta-6 than the benign tumors. ACCs showed significantly higher expression of beta-6 than PAs (p=0.04). No significant difference was observed between ACCs and PLGAs. beta-6 expression was rarely seen in normal salivary gland epithelium and was occasionally present in mucosa overlying the tumors. PAs were high-expressors of tenascin-C with a significant difference relative to ACCs (p=0.03). A majority of tumors in all three tumor types showed high expression of MMP1 with expression significantly greater in the PAs compared to ACCs (p=0.008). We conclude that ACCs and PLGAs express beta-6, tenascin-C, and MMP-1, but that their expression patterns are not significantly different. beta-6 appears to be more closely associated with the malignant tumors, and MMP-1 more closely associated with the benign tumors. We believe that beta-6, tenascin-C, and MMP-1 proteins are part of the molecular repertoire used by salivary tumors for malignant invasion and benign tumor expansion.

Tenascin and beta 6 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.

Oral mucosal carcinogenesis in SENCAR mice.

Animal models of oral carcinogenesis have been developed but most use the hamster buccal pouch or rat oral mucosa. With completion of human and murine genome sequencing, the development of a mouse model of oral carcinogenesis may prove useful for future genomic studies of oral carcinogenesis. To achieve this objective, 30 SENCAR mice were initiated by brush application of palatal, buccal and tongue mucosa with 200 nmol 7,12-dimethylbenz[a]anthracene (DMBA) using 3 treatment regimens, and promoted by brush application with 5 nmol 12-O-tetradecanoylphorbol-13-acetate (TPA) for a total of 28 weeks. Alternatively, 5 mice were treated with 0.5% 4-nitroquinoline-1-oxide (4NQO) alone by brush application for 28 weeks. There were another 6 control mice treated with vehicle alone. The tumor samples were analyzed for the presence of H-ras codon 61 gene mutations using a mutant-allele-specific amplification-polymerase chain reaction (MASA-PCR) technique. The results showed that among the group of 24 mice initiated with DMBA for 2 or 6 weeks, a range of papilliferous lesions were seen on the buccal mucosa comprising papillomas, papillomas with dysplasia and 7 squamous cell carcinomas (SCC). In those 6 mice initiated with 1 week of DMBA, only papillomas developed. In the 5 mice treated with 4NQO, one developed papillomas with dysplasia and two had SCCs in the tongue mucosa but not the buccal mucosa. Both carcinogens induced codon 61 mutation of the H-ras gene at a high frequency. The results indicated that DMBA/TPA and 4NQO in SENCAR mice reliably produced preneoplastic and malignant oral cavity lesions, which resemble the multistages for human oral carcinogenesis, and targeted to site-specific zones of the oral mucosa, namely the buccal mucosa and tongue, respectively. These results show that SENCAR mice can be used as a unique model of oral carcinogenesis with the potential for detailed molecular studies of neoplastic progression to SCC.

Oral spindle cell neoplasms: a review of 307 cases.

The infrequent exposure of pathologists to soft tissue spindle cell neoplasms coupled with overlapping histologic patterns can often make diagnosis challenging. We reviewed all nonodontogenic spindle cell neoplasms seen between 1982 and 2002 (86,162 total accessions). Diagnoses were reclassified according to current standards supplemented with immunohistochemistry. Of the 307 neoplasms reviewed (0.36% of total accessions), neural tumors were the most common benign entities, accounting for 21% of total cases. Kaposi's sarcoma was the most common malignancy, accounting for 67% of all cases. Diagnoses were revised for 57 cases. Schwannoma and neurofibroma were most commonly revised to palisaded encapsulated neuroma. There were 8 myofibromas and 1 inflammatory myofibroblastic tumor. There were no oral leiomyomas; that is, all 4 originally reported cases were reclassified as myofibroma, palisaded encapsulated neuroma, and solitary fibrous tumor. With the exception of Kaposi's sarcoma, oral soft tissue sarcomas were rare; most benign lesions were neural in origin. The relatively high prevalence of some tumors, such as myofibroma, likely reflects the use of immunohistochemistry in the diagnosis of spindle cell tumors.

Oral lesions as the first clinical sign of microscopic polyangiitis: a case report.

Microscopic polyangiitis (MPA) is defined as a systemic necrotizing vasculitis that clinically and histologically affects small-sized vessels without granulomata. The main clinical feature of MPA is renal involvement characterized by rapidly progressing glomerulonephritis. We report a case of oral lesions, initially thought to be associated with Wegener's granulomatosis, as the first sign of MPA in an otherwise healthy man. To our knowledge, this is the first published case of MPA-associated oral lesions.

Assessment of p63 expression in oral squamous cell carcinomas and dysplasias.

OBJECTIVES: p63, a p53 homologue, may be associated with tumorigenesis in epithelial tissues through its inhibition of p53 transactivation functions. We sought to determine the pattern and levels of p63 expression in oral dysplasias and carcinomas using standard immunohistochemical staining. We also assessed and compared expression of p53 and a cell proliferation marker in these lesions. STUDY DESIGN: This retrospective cross-sectional survey (n=67) included hyperkeratosis (10), mild dysplasia (9), moderate dysplasia (11), severe dysplasia/in situ carcinoma (10), squamous cell carcinoma (SCC) (22 [9 well differentiated, 7 moderately differentiated, 6 poor differentiated]), and normal mucosa (5). Serial sections were stained immunohistochemically with antibodies to p63 (4A4 recognizing all p63 isotypes), p53 (DO-7), and Ki-67 (MIB-1) proteins. In preinvasive lesions, both the percentage of positive cells and staining patterns (negative, basal, suprabasal) were assessed. In oral SCCs, the percentage of positive cells was assessed. Statistical analysis was done using the Tukey-Kramer multiple comparisons test. RESULTS: A suprabasal p63 staining pattern was evident in keratinocyte nuclei in the entire range of noninvasive lesions studied, including normal mucosa. Most nuclei in invasive SCCs stained positive. When all grades of dysplasia were combined, the percent of p63 positive cells was significantly greater than hyperkeratosis (P < .01), and well-differentiated SCC (P < .001). Moderately differentiated SCC had statistically significant more positive cells than well-differentiated SSC (P < .01). Comparison of serial sections showed different p63 staining patterns compared to p53 or Ki-67 staining patterns. CONCLUSIONS: We conclude that p63 is expressed in oral carcinomas and dysplasias, as determined by immunohistochemical staining with a primary antibody to all isotypes. Neither staining pattern nor percentage of stained cells could be used to differentiate the lesions studied. The statistically significant differences found between some groups are not likely to be of diagnostic value. p63 is not coexpressed with p53 expression or Ki-67 suggesting functional independence. When antibodies to the p63 isotypes become available, oral dysplasias and carcinomas should be reassessed.

Advanced diagnostic methods in oral and maxillofacial pathology. Part II: immunohistochemical and immunofluorescent methods.

The practice of pathology is currently undergoing significant change, in large part due to advances in the analysis of DNA, RNA, and proteins in tissues. These advances have permitted improved biologic insights into many developmental, inflammatory, metabolic, infectious, and neoplastic diseases. Moreover, molecular analysis has also led to improvements in the accuracy of disease diagnosis and classification. It is likely that, in the future, these methods will increasingly enter into the day-to-day diagnosis and management of patients. The pathologist will continue to play a fundamental role in diagnosis and will likely be in a pivotal position to guide the implementation and interpretation of these tests as they move from the research laboratory into diagnostic pathology. The purpose of this 2-part series is to provide an overview of the principles and applications of current molecular biologic and immunologic tests. In Part I, the biologic fundamentals of DNA, RNA, and proteins and methods that are currently available or likely to become available to the pathologist in the next several years for their isolation and analysis in tissue biopsies were discussed. In Part II, advances in immunohistochemistry and immunofluorescence methods and their application to modern diagnostic pathology are reviewed.

Proliferation and invasion factors in HIV-associated dysplastic and nondysplastic oral warts and in oral squamous cell carcinoma: an immunohistochemical and RT-PCR evaluation.

OBJECTIVE: Oral warts arising in human immunodeficiency virus (HIV) infection occasionally show marked epithelial dysplasia. However, anecdotal evidence suggests that they do not progress to oral squamous cell carcinoma (SCC). Therefore, we evaluated lesions for expression of proteins (tenascin-C, beta6 integrin, and matrix metalloproteinase-1[MMP1]) that have been identified as important in the invasive phase of oral SCC. STUDY DESIGN: Twenty-two oral dysplastic warts from 22 patients and 5 oral SCCs were stained for human papillomavirus (HPV) antigen, proliferation protein Ki-67, tenascin-C, beta6, and MMP1 by immunohistochemical methods. For comparison, 5 nondysplastic warts each from HIV-positive and HIV-negative patients and 5 normal mucosa specimens were included. Sections were semiquantitatively assessed, and results were compared. Because MMP1 was the lowest or least expressed interface protein, MMP1 mRNA was quantitatively assessed from formalin-fixed paraffin-embedded tissue in selected cases with quantitative reverse transcription-polymerase chain reaction. RESULTS: Twenty of 22 dysplastic warts stained positive for human papillomavirus common antigen, and all warts showed high proliferative fractions similar to SCCs. Tenascin-C and beta6 were variably expressed by the dysplastic warts but were consistently expressed at high levels in the SCCs. MMP1 protein levels were negative or low in 20 of 22 in dysplastic warts, but were elevated in 4 of 5 SCCs. MMP1 mRNA analysis indicated that message was low in 4 dysplastic warts and also suggested that protein translation was incomplete in 3 of the warts. CONCLUSION: We conclude that invasion-associated proteins are underexpressed in oral dysplastic warts in HIV-positive men. However, until these patients are followed for extended periods, the risk of development of SCC from oral dysplastic warts remains unknown.

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.

The expression of apoptotic proteins and matrix metalloproteinases in odontogenic myxomas.

PURPOSE: The odontogenic myxoma is a rare benign tumor affecting the jaws. We hypothesize that odontogenic myxomas have dysregulated antiapoptotic mechanisms to assist in neoplastic growth. We believe that antiapoptotic proteins of the Bcl-2 family are over expressed and that tumor cells must generate some form of matrix proteinase. The aim of this study was to evaluate odontogenic myxomas for the expression of cell cycle protein Ki-67, apoptosis-regulating proteins Bcl-2, Bcl-XL, Bak, and Bax, and matrix metalloproteinases MMP-2, MMP-3, and MMP-9. MATERIALS AND METHODS: Odontogenic myxomas submitted to oral pathology between 1974 and 1998 were evaluated. Twenty-six paraffin-embedded tissue sections were used in a standard immunohistochemistry protocol and incubated with one of the following antibodies: Bcl-2, Bcl-XL, Bak, Bax, or Ki-67. The sections were then incubated with anti-immunoglobulin conjugated to peroxidase-labeled dextran polymer in a Tris-HCl buffer. Counts of positive (staining) cells were completed in 5 high-power fields for each specimen. Each slide was reviewed by 2 investigators, and final data were pooled and averaged. RESULTS: Specimen slides showed an increase in cells staining positively for anti-apoptotic proteins Bcl-2 and Bcl-X. An average of 6.5% of specimen cells were positive for Bcl-2 and 10.4% for Bcl-X. Control tissue showed only 1.1% of cells to be positive for Bcl-2 and 1.2% for Bcl-X. Less than 1% of both specimen and control cells stained positively for Ki-67. Proapoptotic proteins (Bak and Bax) were not detected in tumor cells. Ninety percent of tumor cells stained positively for MMP-2 compared with 10% of controls. Specimen and controls were negative for MMP-3 and MMP-9. CONCLUSION: Odontogenic myxoma tumor cells did not show an increase in cell division. Less than 1% of tumor and control cells were positive for Ki-67. Odontogenic myxoma tumor cells showed increased expression of antiapoptotic proteins (Bcl-2 and Bcl-X) and the matrix metalloproteinase MMP-2. This study suggests that 2 mechanisms of disease progression used by the odontogenic myxoma are the production of antiapoptotic proteins and the secretion of matrix metalloproteinases.

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.