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Oral Abstract Session 14 Maxillofacial Pathology And Medicine
Oral Abstract Session 14 MAXILLOFACIAL PATHOLOGY AND MEDICINE Saturday, September 23, 2000, 4:00 pm - 5:30 pm
Functional Activities of P450 1A1, Glutathione-S-Transferase, and Glutathione Levels in Oral Keratinocytes D. Rothas, Ohio State University, College of Dentistry, Postle Hall, Department of Oral and Maxillofacial Surgery, 305 W 12th Ave, Columbus, OH 43210 (Rinaldi A, Morse M, Mallery S) Although the oral cavity’s ability to metabolize chemical carcinogens has been shown empirically by clinical data (association between smoking/alcohol use and oral squamous cell carcinoma [SCC]), little is known about what specific enzymes are involved in carcinogen metabolism within the oral mucosa. Before devising an oral cancer chemopreventive strategy, it is essential to establish that the targeted pathways are present in the oral keratinocytes. Studies in our laboratory have confirmed that the characterized human oral SCC cell cultures (ATCC, Rockville, MD) retain many characteristics of normal oral mucosal tissue, specifically the capacity for carcinogen metabolism. Using cultured SCC cells, this study investigated the effects of curcumin, a recognized chemopreventive agent, on 1) the key carcinogen metabolizing P450 enzyme (1A1) via determination of ethoxyresorufin Odeethylation (EROD) (HPLC fluorescent assay); 2) cellular levels of the cytoprotective tripeptide, glutathione (GSH) (spectrophotometric assay); and 3) functional activities of a key carcinogen-inactivating enzyme (glutathione-S-transferase [GST] isoforms) using 1-chloro-2,3nitrobenzene (CDNB). Because dose-response studies showed that 5 µmol/L curcumin was well tolerated (comparable viabilities [trypan blue exclusion], cell numbers, and protein levels [Lowry] relative to nontreated cultures), 5 µmol/L curcumin in 0.1% DMSO was used for subsequent studies. Metabolism experimental groups (all contained 10 µmol/L ethoxyresorufin [ER]) comprised 1) ER degradation control: medium ⫹ ER ⫹ DMSO; 2) vehicle control: cells ⫹ DMSO; 3) cells ⫹ ER ⫹ DMSO; 4) cells ⫹ curcumin added concurrently with ER; 5) cells ⫹ curcumin (added 72 hours before experiment, medium changed daily) ⫹ ER. EROD results (expressed as % change relative to group 3 cell metabolism ⫾ SD, n ⫽ 4 for all groups) showed comparable EROD after concurrent curcumin delivery (group 4, ⫺2.18 ⫾ 9.05%), whereas sustained curcumin significantly increased EROD (group 5, ⫹89.67 ⫾ 24.50) (P ⬍ .001 3 vs 5, ANOVA followed by multiple t-test). Treatment with 5 µmol/L
82
curcumin significantly increased GSH levels over control (% change GSH levels nmol/mg protein, n ⫽ 12 ⫾ SD, 33.95 ⫾ 46.64) (P ⬍ .05, chi-square one-way classification). Furthermore, the GST activities after culture with or without 5 µmol/L curcumin were 16.06 ⫾ 11.09 and 16.64 ⫾ 8.86, respectively (n ⫽ 12 for both groups, 1 unit conjugates 1.0 mmol of CDNB with reduced GSH per minute at pH 6.5/25°C). These results show that these cell lines retain expression and functional activity of the carcinogen-metabolizing enzymes P450 1A1, GST, and cofactor GSH. The increased ER metabolism noted during sustained curcumin exposure may reflect increased expression or efficiency of 1A1. The increase in the oxidant scavenger GSH may represent GSH sparing in the presence of the antioxidant curcumin. Finally, the GST levels in the oral mucosa are significantly higher than in other tissues (57 times greater than colon cancer cell lines), supporting oral mucosa as a key site for carcinogen metabolism.
References Khafif A, Schantz SP, Chou TC, et al: Quantitation of chemopreventive synergism between (⫺)-epigallocatechin-3-gallate and curcumin in normal, premalignant and malignant human oral epithelial cells. Carcinogenesis 19:419, 1998 Beaumont PO, Moore MJ, Ahmad K, et al: Role of glutathione S-transferases in the resistance of human colon cancer cell lines to doxorubicin. Cancer Res 58:947, 1998
Funding Source: OMFS Student Research Training Award, NIH/ NIDCR DE PO1-12704
Adeno-Associated Virus-MyoD1 Activation of Utrophin in mdx Mouse Muscle G. Bruno, Ohio State University College of Dentistry, Postle Hall, Department of Oral and Maxillofacial Surgery, 305 W 12th Ave, Columbus, OH 43210 (Rath E, Johnson P, Burghes A) Duchenne’s Muscular Dystrophy (DMD) is an X-linked disorder caused by mutations in the dystrophin gene that result in a nonfunctional protein. Clinical manifestations in DMD patients begin in childhood and are characterized by widespread, progressive muscle weakness and wasting, including the orofacial masticatory and perioral musculature. An animal model for DMD is the mdx
AAOMS
•
2000
Oral Abstract Session 14: Maxillofacial Pathology and Medicine mouse that likewise contains a disrupted dystrophin gene and protein. Utrophin, a muscle protein similar to dystrophin, has been used previously in mdx mice to functionally compensate for the dystrophin deficiency. However, the utrophin gene is too large to be inserted into the recombinant adeno-associated virus (rAAV) vector that may be used in gene transfer/therapy protocols. In an attempt to stimulate utrophin expression, an rAAV construct has been made containing the MyoD1 musclespecific transcription factor. We hypothesize that injection of rAAV-MyoD1 will result in an increase in expression of the utrophin gene. Gene transfer of MyoD1 may be a promising approach in the correction/compensation for defects in dystrophin in mdx mice (and humans with DMD). Purpose: The objective of this project is to evaluate the potential use of an rAAV-MyoD1 recombinant construct to stimulate increased levels of gene expression and protein production of the compensatory/redundant utrophin muscle protein in the mdx mouse. Methods: Quadriceps muscles were removed from mdx mice that were injected with 50 µL of the rAAVMyoD1 construct (109 infectious units/mL) in either the right or left quadriceps. The contralateral (nontreated) muscle tissue was used as a control. Immunohistochemistry was performed by standard protocol to examine the levels of the MyoD1 and the utrophin proteins in muscle sections. Either a rabbit polyclonal utrophin or MyoD1 primary antibody was used along with a secondary FITCor TRITC-conjugated donkey anti-rabbit antibody. Slides were evaluated by using a fluorescent microscope. Results: One of four animals treated with the rAAVMyoD1 construct displayed increased levels of MyoD1 and utrophin in the injected muscle. MyoD1 staining in this animal was concentrated in the nuclei and utrophin staining was limited to the endomysium of muscle fibers. MyoD1 protein was not detected, and utrophin protein was less in contralateral control tissues. Increased MyoD1 and utrophin protein levels were difficult to distinguish from background in the remaining animals. Discussion/Conclusions: Our results show successful rAAV-mediated gene transfer of the MyoD1 musclespecific transcription factor into muscle of mdx mice, along with an increase in levels of the utrophin protein. The efficiency of gene transfer was low in our study and might be attributed to lower than desirable titer levels of the rAAV-MyoD1 construct that was used for injection. Further investigation to augment utrophin expression will include increasing the titer of rAAV-MyoD1 that will be injected into mdx muscle. In addition, we plan to evaluate levels of the utrophin protein in the orofacial musculature of mdx mice. These studies may lead, in the future, to potential gene therapeutic treatment of human DMD patients.
AAOMS
• 2000
References Kiliardis S, et al: The effects of myotonic dystrophy and Duchenne muscular dystrophy on the orofacial and dentofacial morphology. Acta Odontol Scand 56:369, 1998 Tinsley JM, et al: Amelioration of the dystrophic phenotype of mdx mice using truncated utrophin transgene. Nature 384:349, 1996
Funding Source: Supported by The Ohio State University and by the Oral and Maxillofacial Surgery Foundation 1999 Student Research Training Award.
3D-SPECT Reconstruction to Assess Bone Invasion by Intraoral Carcinoma Ronald Schimming, MD, DMD, Department of Oral and Maxillofacial Surgery, University Clinics of Freiburg, Hugstetterstr 55, Freiburg, D-79106 Germany (Juengling F, Lauer G, Schmelzeisen R) Purpose: No investigation has proved accurate and reliable for assessing mandibular invasion by carcinoma before surgery. This prospective study was designed to compare a new imaging modality, computer-aided 3D 99mTc-DPD-SPECT reconstruction, with clinical examination, panoramic radiography, computed tomography (CT) scan, and conventional 99mTc-DPD-SPECT investigation in the assessment of mandibular bone invasion by squamous cell carcinoma. Methods: Between January 1997 and December 1999, 88 patients with SCC of the mandibular region were enrolled in this study. In 50 cases, mandibular resection (segmental or marginal) was performed based on the pretherapeutical diagnostic results. Resected mandibles were decalcified and examined for tumor invasion. Imaging studies were read independently by 4 experienced observers. Results: No differences could be found between 3D 99mTc-DPD-SPECT reconstruction and conventional 99mTc-DPD-SPECT investigation. Both techniques showed the highest sensitivity (100%), whereas CT scan showed the highest specificity (93.8%). SPECT investigation had a specificity of 91.6% and the highest efficiency (95.4%). The highest predictive positive value was found for CT scan (92.3%). Clinical examination and panoramic radiography displayed the lowest sensitivity, 82.5% versus 85.0%, and specificity, 79.2% versus 89.5%. Conclusion: This investigation does not provide evidence that 3D 99mTc-DPD-SPECT reconstruction shows advantages compared with conventional 99mTc-DPDSPECT investigation in the assessment of mandibular invasion by SCC. Despite a sensitivity of 100%, the specificity is still in need of improvement. Until newer methods or techniques become available, the combination of CT scan and conventional 99mTc-DPD-SPECT investigation appears to be the best means of detecting
83
Oral Abstract Session 14: Maxillofacial Pathology and Medicine tumor invasion and dictating the appropriate surgical procedure.
Minor Salivary Gland Tumor From the University of Maryland Experience Pornchai Jansisyanont, DDS, University of Maryland, Department of Oral and Maxillofacial Surgery, 666 W Baltimore St, 3-G-21, Baltimore, MD 21201 (Blanchaert R, Ord R) Purpose: Minor salivary gland tumors of the oral cavity are seldom reported separately from other salivary gland tumors. This makes interpretation of the results of such studies in relation to minor salivary gland tumors specifically difficult. This study evaluates the incidence, epidemiology, histology, and treatment of patients with minor salivary gland tumors who presented to the OMS Department at the University of Maryland Medical System. Materials and Methods: The design of the study was the retrospective review of the charts of 58 patients (oral maxillofacial surgery department). Result: Fifty-eight patients with minor salivary gland tumors were identified. Benign minor salivary gland tumors were 22% (13 of 58) and malignant tumors were 78% (45 of 58). There was an overall female preponderance (1.41 to 1). The mean age for malignant minor salivary gland is 5.8 years younger than those with benign. Seventy-six percent were white, and 22% were black. The palate was the most common site for all minor salivary gland tumors (48%). Sixty-one percent of the tumors that occurred in the palate were malignant. Pleomorphic adenoma was the most common minor salivary gland tumor (84.6%) and accounted for 19% of all minor salivary gland tumors. Mucoepidermoid carcinoma was the majority of the malignant salivary gland tumor (48.9%), accounting for 38% of all tumors. Surgery was the sole treatment modality for most of these tumors (71%). Adjunctive therapies with radiation or chemotherapy were identified in 29% of cases. Discussion: The incidence of malignancy identified by this study (78%) is similar to the report of a well-known surgical cancer center and quite different from other reported studies. The palate is the reported site for approximately 40% to 50% of minor salivary gland tumors, with an approximate 50% incidence of malignancy in most studies. Similar to other reports, the palate was the site for 48% of the minor salivary gland tumors; however, the incidence of malignancy was increased at 61%. Other major differences in the findings of this study are the low incidence of pleomorphic adenoma (19% this report and 50% others) and the preponderance of mucoepidermoid carcinoma in this report (29%, 38% this study). These differences are likely because of selection bias, as the University of Maryland is a referral center for oral cancer. 84
References Spiro RH: Salivary neoplasm: Overview of a 35-year experience with 2807 patients. Head and Neck 8:177, 1986 Waldron CA, El-Mofty K, Gnepp DR: Tumors of the intraoral minor salivary glands: A demographic and histologic study of 426 cases. Oral Surg Oral Med Oral Pathol 66:323, 1988 Rivera-Bastidas H, Ocanto RA, Acevedo AM: Intraoral minor salivary gland tumors: A retrospective study of 62 cases in a Venezuelan population. J Oral Pathol Med 25:1, 1996
Funding Source: University of Maryland Oral-Maxillofacial Surgery Department.
Mandibulotomy, a Surgical Approach for Ablation of Oral Cancer: Complications and Contributing Factors Seong-Kyu Byun, DDS, MsD, Ewha Womans University, Department of Oral and Maxillofacial Surgery, Mokdong Hospital 911-1, Mokdong, Yangcheon ku, Seoul, 158-710 South Korea (Choi E, Cha I, Kim M) Purpose: Although some approaches have been introduced for ablative surgery of oral cancer, it has been difficult to get a direct access to oral cavity and oropharynx. Mandibulotomy provides a wide surgical field and ease for complete ablation, but it has been considered a complicated procedure because of postoperative disability and not a few severe complications such as nonunion or osteoradionecrosis. The goal of this study was to analyze complications related with mandibulotomy and its contributing factors. Materials and Methods: We retrospectively studied the 56 patients (51 male, 5 female) who have undergone mandibulotomy at Yonsei medical center over the period from 1989 to 1999. Orthopantomographic, standard periapical radiograph was available before surgery, immediately postoperative, and at the latest follow-up. The range of follow-up periods was 18 to 122 months. The patients were classified according to the following parameters: site of primary tumor, type of osteotomy, site of osteotomy, fixation methods, extraction of adjacent tooth, adjunctive treatments such as preoperative or postoperative radiation therapy, and complications. Results: Complications occurred in 16 patients (28.6%) and are classified to 2 categories, intraoperative and postoperative complications. Nonunion and osteoradionecrosis (ORN) occurred in 5 patients each and were classified as late postoperative complications. The presence of preoperative radiation therapy was the most important factor in the development of nonunion and osteoradionecrosis. Conclusions: The findings of this study suggest the benefits of anterior paramedian mandibulotomy as a surgical approach to oral cancer. Paramedian osteotomy was recommended for preservation of inferior alveolar neurovascular bundle and easiness of surgical access. AAOMS
•
2000
Oral Abstract Session 14: Maxillofacial Pathology and Medicine Osteotomy on anterior mandible, which lies outside of the usual portals of radiation therapy, will decrease the incidence of osteoradioanecrosis. References Shah JP, Kumarswamy SV, Kulkarni V: Comparative evaluation of fixation methods after mandibulotomy for oropharyngeal tumours. Am J Surg 166:431, 1993 Spiro R, Gerold FP, Strong EW: Mandibular ‘‘Swing’’ approach for oral and oropharyngeal tumours. Head Neck Surg 3:371, 1981
Funding Source: Yonsei University Research Fund.
Oral Cancer Molecular Profiling Using High Throughput Analysis Randy Todd, DMD, MD DMSc, Harvard/Massachusetts General Hospital, 188 Longwood Ave, Boston, MA 02115 (Ohyama H, Alevizos I, Khono Y, Warrington J, Wong D) Purpose: Biologically based diagnostic and therapeutic strategies will likely be based on a sound understanding of multiple (rather than individual) genetic events during oral carcinogenesis. This study aims to detail a technique to generate target sample from laser capture microdissection (LCM) human normal and malignant oral epithelium suitable to hybridize high-density oligonucleotide arrays for gene expression profiling. Methods and Materials: Five paired human normal and malignant oral epithelial specimens were snap frozen. RNA was successfully isolated from LCM-isolated tissues using 2 rounds of T7 RNA polymerase linear amplification. The quality and representation of the cRNA were determined using reverse transcription polymerase chain
AAOMS
• 2000
reaction (RT-PCR) and human test 1 GeneChipR probe arrays. Gene expression analysis was performed using HuGenFL GeneChipR probe arrays. Results: LCM was used to microdissect approximately 120,000 cells (28,000 LCM pulses at 30-µm diameter), which yielded ⬃450 ng of RNA. Reverse transcription was performed on 75 ng of isolated RNA, followed by two rounds of T7 RNA polymerase amplification and produced ⬃5 µg of cDNA. RT-PCR of five cellular maintenance transcripts and biotinylated sample hybridization to the human Test 1 GeneChipR probe arrays confirmed that the cRNA from LCM tissues is of sufficient quality and integrity to warrant further analysis. Subsequent hybridization of the sample to the HuGenFL GeneChipR probe array revealed that 23% of the ⬃7,000 represented genes are expressed. Preliminary bioinformatics of the generated databases will be discussed. Conclusion: These results demonstrate that LCMgenerated tissues can generate sufficient quality cRNA for solid phase cDNA microarray analysis, a critical step to determine comprehensive gene expression profiling of human oral carcinogenesis using this high throughput technology. References Luo L, Salunga RC, Guo H, et al: Gene expression profiles of laser-captured adjacent neuronal subtypes. Nature Med 5:117, 1999 Simone NL, Bonner RF, Gillespie JW, et al: Laser-capture microdissection: Opening the microscopic frontier to molecular analysis. Trends Genet 14:272, 1998
Funding Source: PHS Grants P01 DE12467 (D.W.), K02 DE00456 (R.T.), R29 DE11983 (R.T.), the Funds for Discovery (D.W./R.T.), Harvard University Milton Grant (R.T.), and Research Support Grant from the Oral and Maxillofacial Surgery Foundation (O.H./R.T.).
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Functional Activities of P450 1A1, Glutathione-S-Transferase, and Glutathione Levels in Oral Keratinocytes D. Rothas, Ohio State University, College of Dentistry, Postle Hall, Department of Oral and Maxillofacial Surgery, 305 W 12th Ave, Columbus, OH 43210 (Rinaldi A, Morse M, Mallery S) Although the oral cavity’s ability to metabolize chemical carcinogens has been shown empirically by clinical data (association between smoking/alcohol use and oral squamous cell carcinoma [SCC]), little is known about what specific enzymes are involved in carcinogen metabolism within the oral mucosa. Before devising an oral cancer chemopreventive strategy, it is essential to establish that the targeted pathways are present in the oral keratinocytes. Studies in our laboratory have confirmed that the characterized human oral SCC cell cultures (ATCC, Rockville, MD) retain many characteristics of normal oral mucosal tissue, specifically the capacity for carcinogen metabolism. Using cultured SCC cells, this study investigated the effects of curcumin, a recognized chemopreventive agent, on 1) the key carcinogen metabolizing P450 enzyme (1A1) via determination of ethoxyresorufin Odeethylation (EROD) (HPLC fluorescent assay); 2) cellular levels of the cytoprotective tripeptide, glutathione (GSH) (spectrophotometric assay); and 3) functional activities of a key carcinogen-inactivating enzyme (glutathione-S-transferase [GST] isoforms) using 1-chloro-2,3nitrobenzene (CDNB). Because dose-response studies showed that 5 µmol/L curcumin was well tolerated (comparable viabilities [trypan blue exclusion], cell numbers, and protein levels [Lowry] relative to nontreated cultures), 5 µmol/L curcumin in 0.1% DMSO was used for subsequent studies. Metabolism experimental groups (all contained 10 µmol/L ethoxyresorufin [ER]) comprised 1) ER degradation control: medium ⫹ ER ⫹ DMSO; 2) vehicle control: cells ⫹ DMSO; 3) cells ⫹ ER ⫹ DMSO; 4) cells ⫹ curcumin added concurrently with ER; 5) cells ⫹ curcumin (added 72 hours before experiment, medium changed daily) ⫹ ER. EROD results (expressed as % change relative to group 3 cell metabolism ⫾ SD, n ⫽ 4 for all groups) showed comparable EROD after concurrent curcumin delivery (group 4, ⫺2.18 ⫾ 9.05%), whereas sustained curcumin significantly increased EROD (group 5, ⫹89.67 ⫾ 24.50) (P ⬍ .001 3 vs 5, ANOVA followed by multiple t-test). Treatment with 5 µmol/L
82
curcumin significantly increased GSH levels over control (% change GSH levels nmol/mg protein, n ⫽ 12 ⫾ SD, 33.95 ⫾ 46.64) (P ⬍ .05, chi-square one-way classification). Furthermore, the GST activities after culture with or without 5 µmol/L curcumin were 16.06 ⫾ 11.09 and 16.64 ⫾ 8.86, respectively (n ⫽ 12 for both groups, 1 unit conjugates 1.0 mmol of CDNB with reduced GSH per minute at pH 6.5/25°C). These results show that these cell lines retain expression and functional activity of the carcinogen-metabolizing enzymes P450 1A1, GST, and cofactor GSH. The increased ER metabolism noted during sustained curcumin exposure may reflect increased expression or efficiency of 1A1. The increase in the oxidant scavenger GSH may represent GSH sparing in the presence of the antioxidant curcumin. Finally, the GST levels in the oral mucosa are significantly higher than in other tissues (57 times greater than colon cancer cell lines), supporting oral mucosa as a key site for carcinogen metabolism.
References Khafif A, Schantz SP, Chou TC, et al: Quantitation of chemopreventive synergism between (⫺)-epigallocatechin-3-gallate and curcumin in normal, premalignant and malignant human oral epithelial cells. Carcinogenesis 19:419, 1998 Beaumont PO, Moore MJ, Ahmad K, et al: Role of glutathione S-transferases in the resistance of human colon cancer cell lines to doxorubicin. Cancer Res 58:947, 1998
Funding Source: OMFS Student Research Training Award, NIH/ NIDCR DE PO1-12704
Adeno-Associated Virus-MyoD1 Activation of Utrophin in mdx Mouse Muscle G. Bruno, Ohio State University College of Dentistry, Postle Hall, Department of Oral and Maxillofacial Surgery, 305 W 12th Ave, Columbus, OH 43210 (Rath E, Johnson P, Burghes A) Duchenne’s Muscular Dystrophy (DMD) is an X-linked disorder caused by mutations in the dystrophin gene that result in a nonfunctional protein. Clinical manifestations in DMD patients begin in childhood and are characterized by widespread, progressive muscle weakness and wasting, including the orofacial masticatory and perioral musculature. An animal model for DMD is the mdx
AAOMS
•
2000
Oral Abstract Session 14: Maxillofacial Pathology and Medicine mouse that likewise contains a disrupted dystrophin gene and protein. Utrophin, a muscle protein similar to dystrophin, has been used previously in mdx mice to functionally compensate for the dystrophin deficiency. However, the utrophin gene is too large to be inserted into the recombinant adeno-associated virus (rAAV) vector that may be used in gene transfer/therapy protocols. In an attempt to stimulate utrophin expression, an rAAV construct has been made containing the MyoD1 musclespecific transcription factor. We hypothesize that injection of rAAV-MyoD1 will result in an increase in expression of the utrophin gene. Gene transfer of MyoD1 may be a promising approach in the correction/compensation for defects in dystrophin in mdx mice (and humans with DMD). Purpose: The objective of this project is to evaluate the potential use of an rAAV-MyoD1 recombinant construct to stimulate increased levels of gene expression and protein production of the compensatory/redundant utrophin muscle protein in the mdx mouse. Methods: Quadriceps muscles were removed from mdx mice that were injected with 50 µL of the rAAVMyoD1 construct (109 infectious units/mL) in either the right or left quadriceps. The contralateral (nontreated) muscle tissue was used as a control. Immunohistochemistry was performed by standard protocol to examine the levels of the MyoD1 and the utrophin proteins in muscle sections. Either a rabbit polyclonal utrophin or MyoD1 primary antibody was used along with a secondary FITCor TRITC-conjugated donkey anti-rabbit antibody. Slides were evaluated by using a fluorescent microscope. Results: One of four animals treated with the rAAVMyoD1 construct displayed increased levels of MyoD1 and utrophin in the injected muscle. MyoD1 staining in this animal was concentrated in the nuclei and utrophin staining was limited to the endomysium of muscle fibers. MyoD1 protein was not detected, and utrophin protein was less in contralateral control tissues. Increased MyoD1 and utrophin protein levels were difficult to distinguish from background in the remaining animals. Discussion/Conclusions: Our results show successful rAAV-mediated gene transfer of the MyoD1 musclespecific transcription factor into muscle of mdx mice, along with an increase in levels of the utrophin protein. The efficiency of gene transfer was low in our study and might be attributed to lower than desirable titer levels of the rAAV-MyoD1 construct that was used for injection. Further investigation to augment utrophin expression will include increasing the titer of rAAV-MyoD1 that will be injected into mdx muscle. In addition, we plan to evaluate levels of the utrophin protein in the orofacial musculature of mdx mice. These studies may lead, in the future, to potential gene therapeutic treatment of human DMD patients.
AAOMS
• 2000
References Kiliardis S, et al: The effects of myotonic dystrophy and Duchenne muscular dystrophy on the orofacial and dentofacial morphology. Acta Odontol Scand 56:369, 1998 Tinsley JM, et al: Amelioration of the dystrophic phenotype of mdx mice using truncated utrophin transgene. Nature 384:349, 1996
Funding Source: Supported by The Ohio State University and by the Oral and Maxillofacial Surgery Foundation 1999 Student Research Training Award.
3D-SPECT Reconstruction to Assess Bone Invasion by Intraoral Carcinoma Ronald Schimming, MD, DMD, Department of Oral and Maxillofacial Surgery, University Clinics of Freiburg, Hugstetterstr 55, Freiburg, D-79106 Germany (Juengling F, Lauer G, Schmelzeisen R) Purpose: No investigation has proved accurate and reliable for assessing mandibular invasion by carcinoma before surgery. This prospective study was designed to compare a new imaging modality, computer-aided 3D 99mTc-DPD-SPECT reconstruction, with clinical examination, panoramic radiography, computed tomography (CT) scan, and conventional 99mTc-DPD-SPECT investigation in the assessment of mandibular bone invasion by squamous cell carcinoma. Methods: Between January 1997 and December 1999, 88 patients with SCC of the mandibular region were enrolled in this study. In 50 cases, mandibular resection (segmental or marginal) was performed based on the pretherapeutical diagnostic results. Resected mandibles were decalcified and examined for tumor invasion. Imaging studies were read independently by 4 experienced observers. Results: No differences could be found between 3D 99mTc-DPD-SPECT reconstruction and conventional 99mTc-DPD-SPECT investigation. Both techniques showed the highest sensitivity (100%), whereas CT scan showed the highest specificity (93.8%). SPECT investigation had a specificity of 91.6% and the highest efficiency (95.4%). The highest predictive positive value was found for CT scan (92.3%). Clinical examination and panoramic radiography displayed the lowest sensitivity, 82.5% versus 85.0%, and specificity, 79.2% versus 89.5%. Conclusion: This investigation does not provide evidence that 3D 99mTc-DPD-SPECT reconstruction shows advantages compared with conventional 99mTc-DPDSPECT investigation in the assessment of mandibular invasion by SCC. Despite a sensitivity of 100%, the specificity is still in need of improvement. Until newer methods or techniques become available, the combination of CT scan and conventional 99mTc-DPD-SPECT investigation appears to be the best means of detecting
83
Oral Abstract Session 14: Maxillofacial Pathology and Medicine tumor invasion and dictating the appropriate surgical procedure.
Minor Salivary Gland Tumor From the University of Maryland Experience Pornchai Jansisyanont, DDS, University of Maryland, Department of Oral and Maxillofacial Surgery, 666 W Baltimore St, 3-G-21, Baltimore, MD 21201 (Blanchaert R, Ord R) Purpose: Minor salivary gland tumors of the oral cavity are seldom reported separately from other salivary gland tumors. This makes interpretation of the results of such studies in relation to minor salivary gland tumors specifically difficult. This study evaluates the incidence, epidemiology, histology, and treatment of patients with minor salivary gland tumors who presented to the OMS Department at the University of Maryland Medical System. Materials and Methods: The design of the study was the retrospective review of the charts of 58 patients (oral maxillofacial surgery department). Result: Fifty-eight patients with minor salivary gland tumors were identified. Benign minor salivary gland tumors were 22% (13 of 58) and malignant tumors were 78% (45 of 58). There was an overall female preponderance (1.41 to 1). The mean age for malignant minor salivary gland is 5.8 years younger than those with benign. Seventy-six percent were white, and 22% were black. The palate was the most common site for all minor salivary gland tumors (48%). Sixty-one percent of the tumors that occurred in the palate were malignant. Pleomorphic adenoma was the most common minor salivary gland tumor (84.6%) and accounted for 19% of all minor salivary gland tumors. Mucoepidermoid carcinoma was the majority of the malignant salivary gland tumor (48.9%), accounting for 38% of all tumors. Surgery was the sole treatment modality for most of these tumors (71%). Adjunctive therapies with radiation or chemotherapy were identified in 29% of cases. Discussion: The incidence of malignancy identified by this study (78%) is similar to the report of a well-known surgical cancer center and quite different from other reported studies. The palate is the reported site for approximately 40% to 50% of minor salivary gland tumors, with an approximate 50% incidence of malignancy in most studies. Similar to other reports, the palate was the site for 48% of the minor salivary gland tumors; however, the incidence of malignancy was increased at 61%. Other major differences in the findings of this study are the low incidence of pleomorphic adenoma (19% this report and 50% others) and the preponderance of mucoepidermoid carcinoma in this report (29%, 38% this study). These differences are likely because of selection bias, as the University of Maryland is a referral center for oral cancer. 84
References Spiro RH: Salivary neoplasm: Overview of a 35-year experience with 2807 patients. Head and Neck 8:177, 1986 Waldron CA, El-Mofty K, Gnepp DR: Tumors of the intraoral minor salivary glands: A demographic and histologic study of 426 cases. Oral Surg Oral Med Oral Pathol 66:323, 1988 Rivera-Bastidas H, Ocanto RA, Acevedo AM: Intraoral minor salivary gland tumors: A retrospective study of 62 cases in a Venezuelan population. J Oral Pathol Med 25:1, 1996
Funding Source: University of Maryland Oral-Maxillofacial Surgery Department.
Mandibulotomy, a Surgical Approach for Ablation of Oral Cancer: Complications and Contributing Factors Seong-Kyu Byun, DDS, MsD, Ewha Womans University, Department of Oral and Maxillofacial Surgery, Mokdong Hospital 911-1, Mokdong, Yangcheon ku, Seoul, 158-710 South Korea (Choi E, Cha I, Kim M) Purpose: Although some approaches have been introduced for ablative surgery of oral cancer, it has been difficult to get a direct access to oral cavity and oropharynx. Mandibulotomy provides a wide surgical field and ease for complete ablation, but it has been considered a complicated procedure because of postoperative disability and not a few severe complications such as nonunion or osteoradionecrosis. The goal of this study was to analyze complications related with mandibulotomy and its contributing factors. Materials and Methods: We retrospectively studied the 56 patients (51 male, 5 female) who have undergone mandibulotomy at Yonsei medical center over the period from 1989 to 1999. Orthopantomographic, standard periapical radiograph was available before surgery, immediately postoperative, and at the latest follow-up. The range of follow-up periods was 18 to 122 months. The patients were classified according to the following parameters: site of primary tumor, type of osteotomy, site of osteotomy, fixation methods, extraction of adjacent tooth, adjunctive treatments such as preoperative or postoperative radiation therapy, and complications. Results: Complications occurred in 16 patients (28.6%) and are classified to 2 categories, intraoperative and postoperative complications. Nonunion and osteoradionecrosis (ORN) occurred in 5 patients each and were classified as late postoperative complications. The presence of preoperative radiation therapy was the most important factor in the development of nonunion and osteoradionecrosis. Conclusions: The findings of this study suggest the benefits of anterior paramedian mandibulotomy as a surgical approach to oral cancer. Paramedian osteotomy was recommended for preservation of inferior alveolar neurovascular bundle and easiness of surgical access. AAOMS
•
2000
Oral Abstract Session 14: Maxillofacial Pathology and Medicine Osteotomy on anterior mandible, which lies outside of the usual portals of radiation therapy, will decrease the incidence of osteoradioanecrosis. References Shah JP, Kumarswamy SV, Kulkarni V: Comparative evaluation of fixation methods after mandibulotomy for oropharyngeal tumours. Am J Surg 166:431, 1993 Spiro R, Gerold FP, Strong EW: Mandibular ‘‘Swing’’ approach for oral and oropharyngeal tumours. Head Neck Surg 3:371, 1981
Funding Source: Yonsei University Research Fund.
Oral Cancer Molecular Profiling Using High Throughput Analysis Randy Todd, DMD, MD DMSc, Harvard/Massachusetts General Hospital, 188 Longwood Ave, Boston, MA 02115 (Ohyama H, Alevizos I, Khono Y, Warrington J, Wong D) Purpose: Biologically based diagnostic and therapeutic strategies will likely be based on a sound understanding of multiple (rather than individual) genetic events during oral carcinogenesis. This study aims to detail a technique to generate target sample from laser capture microdissection (LCM) human normal and malignant oral epithelium suitable to hybridize high-density oligonucleotide arrays for gene expression profiling. Methods and Materials: Five paired human normal and malignant oral epithelial specimens were snap frozen. RNA was successfully isolated from LCM-isolated tissues using 2 rounds of T7 RNA polymerase linear amplification. The quality and representation of the cRNA were determined using reverse transcription polymerase chain
AAOMS
• 2000
reaction (RT-PCR) and human test 1 GeneChipR probe arrays. Gene expression analysis was performed using HuGenFL GeneChipR probe arrays. Results: LCM was used to microdissect approximately 120,000 cells (28,000 LCM pulses at 30-µm diameter), which yielded ⬃450 ng of RNA. Reverse transcription was performed on 75 ng of isolated RNA, followed by two rounds of T7 RNA polymerase amplification and produced ⬃5 µg of cDNA. RT-PCR of five cellular maintenance transcripts and biotinylated sample hybridization to the human Test 1 GeneChipR probe arrays confirmed that the cRNA from LCM tissues is of sufficient quality and integrity to warrant further analysis. Subsequent hybridization of the sample to the HuGenFL GeneChipR probe array revealed that 23% of the ⬃7,000 represented genes are expressed. Preliminary bioinformatics of the generated databases will be discussed. Conclusion: These results demonstrate that LCMgenerated tissues can generate sufficient quality cRNA for solid phase cDNA microarray analysis, a critical step to determine comprehensive gene expression profiling of human oral carcinogenesis using this high throughput technology. References Luo L, Salunga RC, Guo H, et al: Gene expression profiles of laser-captured adjacent neuronal subtypes. Nature Med 5:117, 1999 Simone NL, Bonner RF, Gillespie JW, et al: Laser-capture microdissection: Opening the microscopic frontier to molecular analysis. Trends Genet 14:272, 1998
Funding Source: PHS Grants P01 DE12467 (D.W.), K02 DE00456 (R.T.), R29 DE11983 (R.T.), the Funds for Discovery (D.W./R.T.), Harvard University Milton Grant (R.T.), and Research Support Grant from the Oral and Maxillofacial Surgery Foundation (O.H./R.T.).
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