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Quantification of P53 in oral epithelial dysplasias via confocal microscopy VS standard microscopy
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY
Abstracts 211
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
Abstracts 211
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