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Multivariate predictors of occult neck metastasis in early oral tongue cancer
P188
Otolaryngology– Head and Neck Surgery August 2003
Research Posters
Problem: Osteogenic protein-1 (OP-1) is a growth factor found to promote osteoblast and chondroblast differentiation of progenitor cells. Dose-dependent increases in OP-1 concentration have been found to increase sulfated glycosaminoglycan synthesis in vitro. In addition, OP-1 administration has been shown to increase type II collagen mRNA synthesis. Much experimentation has been performed in human articular cartilage and has shown increased proteoglycan and type II collagen production. No evidence is available to assess the effect of OP-1 on cartilage production in the trachea in vivo. Methods: We randomly separated 26 New Zealand White rabbits into experimental and control groups. Each rabbit underwent a 2-mm anterior puncture across a tracheal ring (ring 2, 3, or 4). The defect was covered with a collagen sponge with a diameter of 8 mm. The sponge of the control group was treated with 15 L of 5% lactose solution. The sponge of the experimental group was treated with 15 L of OP-1 solution (2.5 mg OP-1 in 1 mL of 5% lactose solution). After 3 weeks, each trachea was harvested and underwent histologic analysis with hematoxylin and eosin, as well as safranin-o. Results: Our presentation will compare and contrast the histologic and gross differences between the control and experimental groups. The clinical implications of these findings and the relationship to tracheal wound healing will also be discussed. Conclusion: Please see results section. Significance: Increased growth of cartilage in response to OP-1 could eventually lead to new methods of promoting tracheal repair. Support: All OP-1 sample was acquired from Dr Koichi Masuda through his research association with Stryker Biotech. Histological analysis was made possible by Dr Williams and his laboratory.
Long-term results of orthotopic tracheal transplantation have not been characterized. The goal of this study was to assess the effect of tracheal allograft re-epithelialization in chronic rejection. Methods: Sixty-four age-matched mice were randomly assigned to four experimental groups. BALB/c donor tracheal segments (5 tracheal rings), were orthotopically transplanted into either syngeneic BALB/c or Class I and Class II MHC mismatched allogeneic C57BL/6 recipients. Groups I and II are both non-immunosuppressed, and tracheas are harvested at 10 days and 100 days, respectively. Groups III and IV are both immunosuppressed with cyclosporin for 50 days and then harvested at 50 days or 100 days, respectively. Tracheal specimens were analyzed with light microscopy and immunohistochemistry, evaluating density of ciliated cells, lumen narrowing, and lymphocytic subpopulations. Results: Group I demonstrated edema of the lamina propria, lymphocytic infiltrate, loss of ciliated epithelia, and narrowing of the airway lumen. Group II demonstrated less than 10% coverage with ciliated epithelial cells. Group III demonstrated a complete layer of ciliated epithelial cells. Group IV demonstrated greater than 70% coverage with ciliated epithelial cells, with comparable density to the continuously immunosuppressed Group III. Conclusion: Orthotopic tracheal allografts maintain ciliated epithelia after immunosuppression has been withdrawn, suggesting that re-epithelialization confers long-term allograft immunotolerance. Significance: Chronic non-specific immunosuppression may not be necessary following tracheal allograft re-epithelialization. Support: None reported.
Long-Term Outcomes of Orthotopic Tracheal Allografts in the Murine Model Houtan Chaboki MD (presenter); Heidi Cleven; Thomas Moran PhD; Lloyd Mayer MD; Eric M Genden MD New York NY; New York NY; New York NY; New York NY; New York NY
Problem: Tracheal transplantation may be a feasible modality to reconstruct extensive, circumferential tracheal defects. Our group has recently demonstrated that immunosuppressed, orthotopically placed tracheal allografts are progressively replaced with recipient-derived epithelium. Preliminary data reveal that once the donor tracheal segment is re-epithelialized with recipient mucosa, immunosuppression can be withdrawn without the risk of acute allograft rejection. Re-epithelialization with host-derived epithelium appears to confer immune tolerance in the acute rejection paradigm.
Multivariate Predictors of Occult Neck Metastasis in Early Oral Tongue Cancer Anthony Michael Sparano MD (presenter); Gregory S Weinstein MD; Ara A Chalian MD; Michael Yodul MD; Randal S Weber MD Philadelphia PA; Philadelphia PA; Philadelphia PA; Philadelphia PA; Philadelphia PA
Problem: The elective dissection of cervical lymph nodes from patients with early oral tongue cancer and a clinically negative neck (T1/T2N0) remains an unsettled issue that continues to be investigated. This study examines clinical and histopathologic factors through univariate and multivariate analysis to correlate the risk of neck micrometastasis in patients with T1/T2N0 squamous cell carcinoma of the oral tongue. Methods: The clinical files and histologic sections of tumor from 45 clinically determined N0 patients were retrospectively analyzed. The factors examined include degree of
POSTERS
R096 R095
Otolaryngology– Head and Neck Surgery Volume 129 Number 2
R097 Tonsillar TⴚCell Subsets in Patients with IgA Nephropathy Hayabusa Nozawa MD (presenter); Miki Takahara MD; Kan Kishibe MD PhD; Satoshi Nonaka MD PhD; Yasuaki Harabuchi MD Hokkaido Japan; Asahikawa Japan; Asahikawa Japan; Asahikawa Japan; Asahikawa-Shi Japan
Problem: IgA nephropathy is related to tonsillar focal infections, and tonsillectomy is known to be effective. Previous studies demonstrated that the area of T-cell nodules was significantly expanded in quantitative immunohistologic analysis on tonsillar tissues from patients with IgA nephropathy. T cells can be divided into CD4 cells and CD8 cells. CD4 cells can be subdivided into CD4⫹CD29⫹ (helper-inducer) cells and CD4⫹CD45RA⫹ (naive or suppressor-inducer) cells. The former were able to proliferate to soluble antigen and provide help for immunoglobulin synthesis, while the latter functioned as inducers of suppression. But, the unique T-cell subsets in IgA nephropathy have not yet been clarified. The purpose of this study is to investigate the T-cell subsets and its functional role in IgA nephropathy. Methods: Lymphocytes were isolated from palatine tonsils by density gradient centrifugation on Ficoll. Two-color flow cytometric analysis was performed for subsets of tonsillar T cells and the expression of immunoglobulin on B cells. Subsets of the CD4 population were determined: CD4⫹CD29⫹ (helper-inducer) cells and CD4⫹CD45RA⫹ (naive or sup-
pressor-inducer) cells. Subsets of the CD8 population were also determined: CD8⫹CD11b dull (suppressor-effector) cells and CD8⫹CD11b bright (cytotoxic-effector/NK) cells. Results: Two-color flow cytometry revealed that the percentage of CD4⫹CD29⫹ cells and surface IgA on B cells was significantly higher in patients with IgA nephropathy than those in patients with recurrent tonsillitis and sleep apnea syndrome. Moreover, the CD45RA⫹ cells percentages of CD4⫹ cells were significantly lower in patients with IgA nephropathy than those in patients with recurrent tonsillitis. We obtained interesting results about some subsets. Conclusion: These results suggest that CD4⫹CD29⫹ cells may increase immunoglobulin synthesis and it may play a key role in the pathogenesis of IgA nephropathy. Significance: It has been proposed that IgA nephropathy results from hyperactivity of the mucosal immune systems including tonsils. T-cell subsets in tonsils may be important factor for control of immune systems of IgA nephropathy. Support: None reported. R098 Identification of Putative HypermethylationInactivated Tumor Suppressor Genes in HNSCC Michael M Kim MD (presenter); Patrick K Ha MD; Zhongmin Guo MD PhD; Nicole Benoit BS; Keishi Yamashita PhD; Joseph Califano MD; David Sidransky MD Baltimore MD; Baltimore MD; Baltimore MD; Baltimore MD; Baltimore MD; Baltimore MD; Baltimore MD
Problem: Hypermethylation of tumor suppressor gene (TSG) promoter regions has been described for a limited number of genes in head and neck squamous cell carcinoma (HNSC). We describe a genome-wide strategy to identify putative tumor suppressor genes in HNSC that are inactivated by promoter methylation. Methods: HNSC cell lines were treated with a demethylating agent to disinhibit expression of putative TSGs. Identification of candidates was based upon significant expression increases compared to untreated controls on cDNA microarrays. Genes downregulated in primary HNSC were identified by comparison of normal tissue and tumors using similar arrays. Downregulated genes common to these groups were verified by RT-PCR and subjected to PCR-based methylation analysis in primary tumors. Results: Demethylating cell lines exhibited expression increases in 364 genes, and primary tumors showed 1106 genes with expression decreases, with 52 genes common to both groups. RT-PCR confirmed increased expression in cell lines for approximately 30% of these genes. Conclusion: Genome-wide screening for promoter hypermethylation-inactivated genes in primary HNSC and cell lines facilitates identification of putative tumor suppressor genes that may be important in HNSC development. Significance: This study demonstrates a powerful method
POSTERS
tumor-cell differentiation, staging, presence of perineural and angiolymphatic invasion, type of invasion front, depth of muscle invasion, and tumor thickness. Results: Independent correlates of positive occult neck metastasis included greater tumor thickness (P ⫽ 0.01), greater depth of muscle invasion (P ⫽ 0.01), T2 stage (P ⫽ 0.01), poorly differentiated tumors (P ⫽ 0.007), infiltratingtype invasion front (P ⫽ 0.03), presence of perineural invasion (P ⫽ 0.001), and presence of angiolymphatic invasion (P ⫽ 0.005). The final multivariate model for estimation of an increased probability of occult neck disease included greater tumor thickness, presence of perineural invasion, infiltratingtype invasion front, poorly differentiated tumors, and T2 stage. Conclusion: The multivariate model derived from this study appears to be a more reliable method for determining the patients most likely to benefit from elective neck dissection. Significance: The clinical and histopathologic factors studied here permit greater selectivity and more informed decision making than does pre-surgical evaluation, when addressing elective neck treatment for early N0 oral tongue cancer. Support: None reported.
Research Posters P189
Otolaryngology– Head and Neck Surgery August 2003
Research Posters
Problem: Osteogenic protein-1 (OP-1) is a growth factor found to promote osteoblast and chondroblast differentiation of progenitor cells. Dose-dependent increases in OP-1 concentration have been found to increase sulfated glycosaminoglycan synthesis in vitro. In addition, OP-1 administration has been shown to increase type II collagen mRNA synthesis. Much experimentation has been performed in human articular cartilage and has shown increased proteoglycan and type II collagen production. No evidence is available to assess the effect of OP-1 on cartilage production in the trachea in vivo. Methods: We randomly separated 26 New Zealand White rabbits into experimental and control groups. Each rabbit underwent a 2-mm anterior puncture across a tracheal ring (ring 2, 3, or 4). The defect was covered with a collagen sponge with a diameter of 8 mm. The sponge of the control group was treated with 15 L of 5% lactose solution. The sponge of the experimental group was treated with 15 L of OP-1 solution (2.5 mg OP-1 in 1 mL of 5% lactose solution). After 3 weeks, each trachea was harvested and underwent histologic analysis with hematoxylin and eosin, as well as safranin-o. Results: Our presentation will compare and contrast the histologic and gross differences between the control and experimental groups. The clinical implications of these findings and the relationship to tracheal wound healing will also be discussed. Conclusion: Please see results section. Significance: Increased growth of cartilage in response to OP-1 could eventually lead to new methods of promoting tracheal repair. Support: All OP-1 sample was acquired from Dr Koichi Masuda through his research association with Stryker Biotech. Histological analysis was made possible by Dr Williams and his laboratory.
Long-term results of orthotopic tracheal transplantation have not been characterized. The goal of this study was to assess the effect of tracheal allograft re-epithelialization in chronic rejection. Methods: Sixty-four age-matched mice were randomly assigned to four experimental groups. BALB/c donor tracheal segments (5 tracheal rings), were orthotopically transplanted into either syngeneic BALB/c or Class I and Class II MHC mismatched allogeneic C57BL/6 recipients. Groups I and II are both non-immunosuppressed, and tracheas are harvested at 10 days and 100 days, respectively. Groups III and IV are both immunosuppressed with cyclosporin for 50 days and then harvested at 50 days or 100 days, respectively. Tracheal specimens were analyzed with light microscopy and immunohistochemistry, evaluating density of ciliated cells, lumen narrowing, and lymphocytic subpopulations. Results: Group I demonstrated edema of the lamina propria, lymphocytic infiltrate, loss of ciliated epithelia, and narrowing of the airway lumen. Group II demonstrated less than 10% coverage with ciliated epithelial cells. Group III demonstrated a complete layer of ciliated epithelial cells. Group IV demonstrated greater than 70% coverage with ciliated epithelial cells, with comparable density to the continuously immunosuppressed Group III. Conclusion: Orthotopic tracheal allografts maintain ciliated epithelia after immunosuppression has been withdrawn, suggesting that re-epithelialization confers long-term allograft immunotolerance. Significance: Chronic non-specific immunosuppression may not be necessary following tracheal allograft re-epithelialization. Support: None reported.
Long-Term Outcomes of Orthotopic Tracheal Allografts in the Murine Model Houtan Chaboki MD (presenter); Heidi Cleven; Thomas Moran PhD; Lloyd Mayer MD; Eric M Genden MD New York NY; New York NY; New York NY; New York NY; New York NY
Problem: Tracheal transplantation may be a feasible modality to reconstruct extensive, circumferential tracheal defects. Our group has recently demonstrated that immunosuppressed, orthotopically placed tracheal allografts are progressively replaced with recipient-derived epithelium. Preliminary data reveal that once the donor tracheal segment is re-epithelialized with recipient mucosa, immunosuppression can be withdrawn without the risk of acute allograft rejection. Re-epithelialization with host-derived epithelium appears to confer immune tolerance in the acute rejection paradigm.
Multivariate Predictors of Occult Neck Metastasis in Early Oral Tongue Cancer Anthony Michael Sparano MD (presenter); Gregory S Weinstein MD; Ara A Chalian MD; Michael Yodul MD; Randal S Weber MD Philadelphia PA; Philadelphia PA; Philadelphia PA; Philadelphia PA; Philadelphia PA
Problem: The elective dissection of cervical lymph nodes from patients with early oral tongue cancer and a clinically negative neck (T1/T2N0) remains an unsettled issue that continues to be investigated. This study examines clinical and histopathologic factors through univariate and multivariate analysis to correlate the risk of neck micrometastasis in patients with T1/T2N0 squamous cell carcinoma of the oral tongue. Methods: The clinical files and histologic sections of tumor from 45 clinically determined N0 patients were retrospectively analyzed. The factors examined include degree of
POSTERS
R096 R095
Otolaryngology– Head and Neck Surgery Volume 129 Number 2
R097 Tonsillar TⴚCell Subsets in Patients with IgA Nephropathy Hayabusa Nozawa MD (presenter); Miki Takahara MD; Kan Kishibe MD PhD; Satoshi Nonaka MD PhD; Yasuaki Harabuchi MD Hokkaido Japan; Asahikawa Japan; Asahikawa Japan; Asahikawa Japan; Asahikawa-Shi Japan
Problem: IgA nephropathy is related to tonsillar focal infections, and tonsillectomy is known to be effective. Previous studies demonstrated that the area of T-cell nodules was significantly expanded in quantitative immunohistologic analysis on tonsillar tissues from patients with IgA nephropathy. T cells can be divided into CD4 cells and CD8 cells. CD4 cells can be subdivided into CD4⫹CD29⫹ (helper-inducer) cells and CD4⫹CD45RA⫹ (naive or suppressor-inducer) cells. The former were able to proliferate to soluble antigen and provide help for immunoglobulin synthesis, while the latter functioned as inducers of suppression. But, the unique T-cell subsets in IgA nephropathy have not yet been clarified. The purpose of this study is to investigate the T-cell subsets and its functional role in IgA nephropathy. Methods: Lymphocytes were isolated from palatine tonsils by density gradient centrifugation on Ficoll. Two-color flow cytometric analysis was performed for subsets of tonsillar T cells and the expression of immunoglobulin on B cells. Subsets of the CD4 population were determined: CD4⫹CD29⫹ (helper-inducer) cells and CD4⫹CD45RA⫹ (naive or sup-
pressor-inducer) cells. Subsets of the CD8 population were also determined: CD8⫹CD11b dull (suppressor-effector) cells and CD8⫹CD11b bright (cytotoxic-effector/NK) cells. Results: Two-color flow cytometry revealed that the percentage of CD4⫹CD29⫹ cells and surface IgA on B cells was significantly higher in patients with IgA nephropathy than those in patients with recurrent tonsillitis and sleep apnea syndrome. Moreover, the CD45RA⫹ cells percentages of CD4⫹ cells were significantly lower in patients with IgA nephropathy than those in patients with recurrent tonsillitis. We obtained interesting results about some subsets. Conclusion: These results suggest that CD4⫹CD29⫹ cells may increase immunoglobulin synthesis and it may play a key role in the pathogenesis of IgA nephropathy. Significance: It has been proposed that IgA nephropathy results from hyperactivity of the mucosal immune systems including tonsils. T-cell subsets in tonsils may be important factor for control of immune systems of IgA nephropathy. Support: None reported. R098 Identification of Putative HypermethylationInactivated Tumor Suppressor Genes in HNSCC Michael M Kim MD (presenter); Patrick K Ha MD; Zhongmin Guo MD PhD; Nicole Benoit BS; Keishi Yamashita PhD; Joseph Califano MD; David Sidransky MD Baltimore MD; Baltimore MD; Baltimore MD; Baltimore MD; Baltimore MD; Baltimore MD; Baltimore MD
Problem: Hypermethylation of tumor suppressor gene (TSG) promoter regions has been described for a limited number of genes in head and neck squamous cell carcinoma (HNSC). We describe a genome-wide strategy to identify putative tumor suppressor genes in HNSC that are inactivated by promoter methylation. Methods: HNSC cell lines were treated with a demethylating agent to disinhibit expression of putative TSGs. Identification of candidates was based upon significant expression increases compared to untreated controls on cDNA microarrays. Genes downregulated in primary HNSC were identified by comparison of normal tissue and tumors using similar arrays. Downregulated genes common to these groups were verified by RT-PCR and subjected to PCR-based methylation analysis in primary tumors. Results: Demethylating cell lines exhibited expression increases in 364 genes, and primary tumors showed 1106 genes with expression decreases, with 52 genes common to both groups. RT-PCR confirmed increased expression in cell lines for approximately 30% of these genes. Conclusion: Genome-wide screening for promoter hypermethylation-inactivated genes in primary HNSC and cell lines facilitates identification of putative tumor suppressor genes that may be important in HNSC development. Significance: This study demonstrates a powerful method
POSTERS
tumor-cell differentiation, staging, presence of perineural and angiolymphatic invasion, type of invasion front, depth of muscle invasion, and tumor thickness. Results: Independent correlates of positive occult neck metastasis included greater tumor thickness (P ⫽ 0.01), greater depth of muscle invasion (P ⫽ 0.01), T2 stage (P ⫽ 0.01), poorly differentiated tumors (P ⫽ 0.007), infiltratingtype invasion front (P ⫽ 0.03), presence of perineural invasion (P ⫽ 0.001), and presence of angiolymphatic invasion (P ⫽ 0.005). The final multivariate model for estimation of an increased probability of occult neck disease included greater tumor thickness, presence of perineural invasion, infiltratingtype invasion front, poorly differentiated tumors, and T2 stage. Conclusion: The multivariate model derived from this study appears to be a more reliable method for determining the patients most likely to benefit from elective neck dissection. Significance: The clinical and histopathologic factors studied here permit greater selectivity and more informed decision making than does pre-surgical evaluation, when addressing elective neck treatment for early N0 oral tongue cancer. Support: None reported.
Research Posters P189