Expression of BCL-2 proto-oncogene in oral epithelial dysplasia and carcinoma: an immunohistochemical study

210 Abstracts

O R A L SURGERY O R A L MEDICINE O R A L P A T H O L O G Y

August 1996 referred to as colloid, hyaline, cytoid, or Civatte bodies. As they display strong immunoreactivity for keratin, they have been interpreted as degenerative keratinocytes. The pathogenesis of LP is believed to involve cell-mediated immunity. D a m a g e to the basal layer appears to involve apoptosis as an early event. Apoptosis, programmed cell death, is a genetically-mediated m e c h a n i s m controlling deletion of individual cells in normal and diseased tissue. It is believed that apoptosis functions to maintain genetic fidelity, minimize phenotypic variation and eliminate genotypic alteration. A n understanding of apoptosis in LP m a y contribute to an understanding of the pathogenesis of the disease. Apoptosis has not been well-characterized in oral LP, therefore, the purpose of the present investigation was to assess its extent in a series of cases. 30 paraffin-embedded cases were retrieved from the files of the Division of Oral, Head & Neck Pathology at Emory University School of Medicine. 16 control cases of focal fibrous hyperplasia were used. Apoptosis was assessed by in situ end labeling of new 3 ' - O H D N A ends generated by D N A fragmentation utilizing the Apop Tag (Oncor, Inc.) detection system. Quantitation was performed by counting the number of labeled cells per high power field, sequentially covering the entire epithelial surface of each section. The mean number of labeled cells per high power field was 0.008 for controls and 2.0 for LP cases. Results were significant for increased apoptosis in LP by Chi square (p < 0.0001). In conclusion, increased apoptosis in LP, demonstrated by in situ endlabeling, supports a hypothesis for the pathogenesis of LP that includes factors, such as secretion of g a m m a interferon by activated lymphocytes, that would cause an increase in differentiation rate.

PHENETHYL ISOTHIOCYANATE(PEIT) INHIBITS NITROSAMINE-MEDIATED CARCINOGENESIS IN HAMSTER BUCCAL POUCH EPITHELIUM(HBPE). Z. Batrouni, *K.-W. Chang, and D.

Solt. Northwestern University Department of Pathology, Chicago, IL, and *Faculty of Dentistry, National Yang-Ming University, Taipei, Taiwan, Republic of China. The purpose of this study was to determine whether PEIT, a vegetable-derived cancer chemopreventive agent inhibits HBPE carcinogenesis mediated by N-methyl-N-benzylnitrosamine(MBN). PEIT(50mM) in corn oil(CO) was topically applied to both buccal pouches of three hamsters(group 1), thrice-weekly for 2 weeks followed by twice-weekly(Monday/Thursday) topical treatments of a mixture of M B N and PEIT(both at 50mM) for 22 weeks. During the 22 weeks of M B N treatment, the group 1 hamsters also received PEIT(50mM) in CO, on Tuesdays and Fridays. Three group 2 hamsters were similarly treated with the vehicle CO, and M B N in CO, during the 24 week experiment. At the completion of the experiment, whereas the group 2(unprotected) hamsters exhibited a total of 60 epithelial neoplasms(23 _+ 10 tumors/hamster), including squamous cell carcinomas, a total of only 4 epithelial neoplasms were observed in the group I(PEIT protected) hamsters pouches. In a follow-up experiment, PEIT(25mM) in CO also strongly inhibited MBN-mediated induction of microscopic epithelial cell loci exhibiting either gamma-glutamyl transpeptidase(GGT) histochemical activity or immunohistochemical staining for the p53 tumor suppressor gene product. These presumptive precancerous foci were identified in HBPE whole mounts prepared after 8 to 13 weeks of topical M B N ( 2 5 m M ) treatment. W e conclude that PEIT i s a potent inhibitor of MBN-mediated HBPE carcinogenesis, and that early G G T and p53 loci are predictive of chemoprevention in this experimental model of oral carcinogenesis.

THE RELATIONSHIP OF P53 AND HPV IN ORAL CARCINOMAIN-SITU. G. Werner and D-J. Summerlin. Indiana U. Indianapo-

lis. Despite numerous advances in etiology and epidemiology, the m e c h a n i s m s involved in oral carcinogenesis remain obscure. Current research provides results which suggest that p53 mutation as well as h u m a n papillomavirus (HPV) infection play a role in the development of oral s q u a m o u s cell carcinoma. The intent of this study was to uncover the relationship of H P V infection to p53 expression in oral carcinoma-in-situ and to examine the c o m m o n histologic characteristics evidenced within each group. Formalinfixed and paraffin-embedded specimens of 32 histologically confirmed cases of carcinoma-in-situ were examined for p53 protein by immunofluorescence with the confocal microscope and for the presence of HPV types 6/11, 16/18 and 31/33/51 infection by D N A in-situ hybridization. The presence of p53 protein was detected in 23 of 32 specimens (72%) including 1 case with coexisting H P V infection. The presence of HPV was detected in 9 of 32 cases (28%). There was one specimen that was unreactive for both H P V and p53. The 32 patients studied were all white with an average age of 57.7 years. 21 of these 32 patients (66%) were men, whereas all 9 of the reported HPV-positive cases were m e n with an average age of 46.3 years. In addition, distinctive histopathologic characteristics were evidenced in the nine reported cases of HPV positivity, including high-level mitotic figures, increased cells per unit area, high-level pleomorphism and highlevel dyskeratotic cells. This study supports the conclusion that HPV infection and p53 mutation represent two separate pathways by which histologi c evidence of carcinoma-in-situ may be produced. The distinct clinicopathologic features also suggest that these pathways m a y lead to differing prognoses of these subsets of carcinoma-in-situ.

EXPRESSION OF BCL-2 PROTO-ONCOGENE IN ORAL EPITHELIAL DYSPLASIA AND CARCINOMA: AN IMMUNOHISTOCHEMICAL STUDY. B. Singh, J. Franklin, B. Whitaker, and F.

Chandler Jr. Medical College of Georgia, Augusta. The rote o f oncogenes and suppressor genes in the initiation and progression of oral epithelial neoplasia is becoming increasingly clear in the recent years. However, data on the recently proposed third category of oncogenes considered to regulate programmed cell death (including bcl-2) are lacking (Korsmeyer, S.J. Blood 80:879, 1992). Therefore the objective of this study was to examine the status of bcl-2 in oral lesions. For this purpose 5g thick sections of formalin fixed tumors from archival paraffin blocks were examined by using immunohistochemical techniques using M A b s (DAKO-Ab124) to bcl-2 oncoprotein. Immunohistochemistry revealed cytoplasmic staining in four (n = 20) mild dysplasias, six (n = 20) moderate dysplasias and 11 (n = 20) severe dysplasias. Bcl-2 immunoreactivity was primarily observed in cells with features of dysplasia whereas the superficial, K-13 positive, differentiating cells showed diffuse, faint, or no reactivity. The dysplastic lesions exhibited varying degrees of staining intensities and percentage of reactive cells. Bcl-2 immunoreactivity was observed in 10 (n = 40) squamous cell carcinomas. Comparatively, a higher percentage of poorly differentiated carcinomas exhibited immunoreactivity that was heterogeneous in all lesions. In comparison all basal cell carcinomas (n = 15) were positive for bcl-2 oncoprotein. In all examined specimens, 10 revealed bcl-2 i m m u noreactivity in the nondysplastic epithelium contiguous to neoplastic epithelium. The sequential and proportional expression of bel-2 in varying degrees of dysplasia; carcinomas and nondysplastic epithelium suggests that bcl-2 expression is an early event

ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY

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Volume 82, Number 2 in oral tumor progression similar to that reported in carcinomas of the cervix and colon. The expression of bcl-2 oncoprotein has not been shown to independently induce cell proliferation or transformation. However it synergizes with other oncogenes, such as myc, in malignant transformations, attesting to its plausible role in oral carcinogenesis. CANCER RISK FACTORS AND HPV FREQUENCY IN CONTROL AND ORAL SQUAMOUS CELL CARCINOMA PATIENTS. K. Summersgill, E. Smith, H. Hoffman, J. Ricks, T. Haugen, and LI Turek. U. of lowa and VAMC, Iowa City. Although the association of h u m a n papillomavirus (HPV) with cervical carcinoma is well established, that with oral squamous cell cancer is less so. The purpose of this study is to compare case and control populations as to potential risk factors. Methods: Control subjects were obtained to match the case population for age and sex. The presence of HPV is determined by polymerase chain reaction (PCR) of oral squamous cells, which were obtained by saline solution rinse. Phenol-extracted D N A underwent TaqStart PCR with H P V consensus M Y 0 9 / M Y 1 1 primers and beta-globin primers. PCR product was transferred to dot blot membranes, which were probed with 3zp-labeled consensus H P V probes. Results: The sample contains 163 controls and 79 cases. Cancer cases are more likely to be current smokers and current alcohol drinkers than are controls. Cancer cases are twice as likely to be HPV-positive than controls (19.0% vs 9.8%). HPV positive cases and controls are disproportionately younger (e.g., 24% of the cancer cases were <55 years old, yet they accounted for 53% of the HPV positive cases). The controls who are HPV positive are more likely to have never smoked and to have never drunk alcohol than controls who are HPV negative. However, the cases who are H P V positive are more likely to be smokers than the cases who are HPV negative. H P V positive and negative cases show little difference in alcohol use status. Future study will include determining HPV types, P C R of oral cancer biopsies, P C R of follow-up oral swishes of cancer patients, and determination of HPV integration in cancers. Conclusions: Smoking, alcohol use, and HPV positivity are associated with increased risk for oral cancer. QUANTIFICATION OF P53 IN ORAL EPITHELIAL DYSPLASIAS VIA CONFOCAL MICROSCOPY VS STANDARD MICROSCOPY. S. Ganatra, D-J. Summerlin, A.H. Kafrawy, C.E. Tomich, M.L. Van Dis, and S.L. Zunt (U. of Nebr. Medical Center, Lincoln and Indiana U., Indianapolis) p53 expression has been shown to be elevated in oral epithelial dysplasias associated with alcohol and tobacco use. The intent of this investigation was twofold: to determine if this association was reproducible and to determine if immunofluorescent testing with confocal microscopy represented a more sensitive and objective analytic tool than standard immunohistochemistry. Sixty-one cases of epithelial dysplasia were obtained from the Indiana University Oral Pathology Biopsy Service and divided into three groups. Group 1 consisted of 19 dysplasias from patients with no habits. Group 2 consisted of 21 dysplasias from patients with a history of smoking without alcohol use. Group 3 consisted of 21 dysplasias from patients with a history of smoking and alcohol consumption. Each case underwent standard immunohistochemical testing with subjective analysis as well as immunofluorescent testing with computer quantification by a confocal laser microscope and associated software. Both techniques were used to quantify intensity of stain and percentage of positive cells (percent area). Immunohistochemical analysis of percent area and stain intensity were not significantly different between the groups.

Immunofluorescent analysis, however, did demonstrate significantly higher intensity (p < 0.03) for group 3 and group 1 than for group 2 but percent area measurements for the three groups were not significantly different. Intensity measurements between the two methods were not correlated (p = 0.22) but percent area measurements were (p <0.01). This study differs from previous studies in that no difference was noted in p53 expression in dysplasia with or without habits. In addition, immunofluorescent testing with confocal microscopy was found to produce significantly different values of percent area and represents a more objective m e c h a n i s m of analysis. EXPRESSION OF MAJOR HISTOCOMPATIBILITY COMPLEX (CLASS I) IN PREMA/IGNANT AND MALIGNANT EPITHELIUM. 1. Bhattacharyya and D-J. Summerlin. U. Nebraska Lincoln and Indiana U. Indianapolis Squamous cell carcinomas (SCC) account for over 90% of oral malignancies and are thought to undergo a multistep neoplastic evolution, involving sequences of genetic alterations leading to a malignant phenotype. One such alteration is thought to involve Major Histocompatibility Complex (MHC) class I molecules. Previous studies have demonstrated that decreased expression of M H C class I antigens correlates with poorer prognosis. M H C class I expression can be determined by the presence beta-2-microglobulin (b2m), as this molecule is bound to M H C class I molecules. In addition, h u m a n papillomavirus (HPV) has been implicated in the development of numerous malignancies, including oral cancer and has been correlated with the expression of M H C class I molecules. However, little has been published on the comparative progression of expression of these entities in premalignancies. The intent of this study was to determine the expression of b2m and HPV in oral premalignancies and malignancies and correlate these results. For this study, 25 cases each of histologically confirmed poorly (PD) and well-differentiated (WD) carcinoma, mild (MD) and severe dysplasia (SD), and normal m u c o s a (NM) were obtained from the archives of the Department of Oral Pathology, Indiana University Medical Center. All samples demonstrated b 2 m positivity whereas only one case of SD was positive for HPV. With confocal microscopy, N M and M D were shown to have statistically significantly higher staining intensity for b 2 m than SD, PD, W D (p < 0.04). N M also had a significantly higher positive staining area than SD, MD, W D and PD (p < 0.01). M D and SD demonstrated higher positive staining than W D and PD (p < 0.01) and W D was significantly higher than PD (p < 0.01). The results of this study confirm that lower levels of M H C class I expression correlate with degree of expression. Also, HPV was not shown to be an important variable in this stud}r group. Further longitudinal studies in regard to prognosis should be performed to cement the possibility that M H C class I expression could predict behavior of oral premalignancies and malignancies. ORAL BASAL CELL CARCINOMA: REPORT OF A CASE WITH IMMUNOHISTOCHEMICAL FINDINGS. I. Koutlas, R. Vickers, E. Rest and P. Gaspard. University of Minnesota, Minneapolis. Basal cell carcinoma (BCC) arising from oral m u c o s a is a controversial or nonentity. Ameloblastoma, peripheral ameloblas~ toma (PA), and basal cell adenoma (BCA) are included in the differential diagnosis of such neoplasms. A possible example of oral B C C is presented that originated on the posterior mandibular m u c o s a and gingiva of a 67-year-old woman. It recurred six times after local excision during a period of 8 years and, most recently extended to include buccal mucosa. Tissue samples of the tumor were evaluated recently with the monoclonal antibody Ber-EP4 (Dako, 1:80) that has been used in