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Genotype-phenotype correlation in a new family with TECTA mutation
Otolaryngology– Head and Neck Surgery Volume 129 Number 2
R035 P53 Mutations in Head and Neck Carcinoma: Correlating Current Detection Methods Thomas R Lowry MD (presenter); Joseph A Brennan MD San Antonio TX; Boerne TX
Problem: To correlate currently accepted methods of p53 mutation detection in head and neck carcinoma with quantitative rapid-cycle, real-time PCR analysis. Methods: Fresh-frozen tissue samples from 37 primary head and neck squamous cell carcinomas were subjected for analysis. Presence of p53 mutation was determined by immunohistochemical staining, genomic DNA sequencing, and quantitative rapid-cycle real-time PCR using the LightCycler. Results were correlated using standard statistical analysis. Results: 21 of 37 samples (57%) showed positive reaction for p53 protein on immunohistochemical staining. Results of genomic DNA sequencing and rapid-cycle, real time PCR using the LightCycler are presented. Conclusion: Traditional methods for detection of p53 mutations in head and neck carcinomas, including immunohistochemistry and genomic DNA sequencing, have certain limitations. p53 quantification using rapid-cycle, realtime PCR is an evolving technology which possesses advantages over these limitations. This study demonstrates that rapid-cycle, real-time PCR is an accurate and efficient method for screening head and neck carcinomas for presence of p53 mutations. Significance: Correlation of methods for p53 mutation detection in head and neck carcinomas. Support: This research is part of a larger study involving p53 analysis and real-time PCR. Total funding to date is from the Surgeon General Office in the amount of $151,000. R036 Evaluation of the Role of the Epstein-Barr Virus in 300 Primary SCCHN David Goldenberg MD (presenter); Nicole Benoit BS; William Westra MD; Wayne M Koch MD; David Sidransky MD; Joseph Califano MD Baltimore MD; Baltimore MD; Baltimore MD; Columbia MD; Baltimore MD; Baltimore MD
Problem: Recently, multiple studies have been published linking EBV with oral squamous cell carcinoma
(OSCC) and, to a lesser extent, hypopharyngeal and laryngeal tumors. These reports have varied in sample size and methodology. We used a sensitive and specific method of detection of EBV, quantitative PCR, in a large cohort of patients with head and neck squamous cell carcinoma (HNSCC) to clarify the possible role of EBV in head and neck cancers and correlate EBV status with exposure history, grade, and p53 status. Methods: We examined 300 head and neck squamous cell carcinomas (HNSCC) for the presence of EBV using quantitative PCR (QPCR) for separate detection of BAM and EBNA sequences in the EBV genome with analysis of beta-actin for quantitation of genomic DNA copy number. 84 larynx, 30 hypopharynx, 71 oropharynx, and 109 oral cavity tumors were analyzed for EBV quantity, expressed as the number of viral copies/genome/cell. Results for EBV were correlated with p53 mutation status, grade, smoking, and alcohol exposure. Results: Only 2 of 300 samples (⬍1%) were overtly positive (defined as ⬎0.1 viral copies/genome/cell). EBV DNA was detected in low levels (defined as ⬎0.01 and ⬍0.1 copies/genome/cell) in 6 of 300 tumors (2%) and in insignificant levels (defined as ⬍0.01 viral copies/genome/cell) in 68 of 300 tumors (23%). No correlation was found between overt or low-level EBV positivity and either smoking exposure, ethanol exposure, p53 status, or tumor grade. Conclusion: In the overwhelming majority of HNSC EBV does not appear to contribute to growth of a dominant clonal population with integrated EBV genome and is unlikely to be etiologic in tumor development. These very low quantities of EBV detected in a minority of HNSC may be related to the presence of EBV genome in rare lymphoid or epithelial cells adjacent to primary HNSC. Significance: This study demonstrates that although EBV presence may be found in low levels in some head and neck SCC, it is unlikely that it plays a role in tumor development. Support: None reported.
R037 Genotype-Phenotype Correlation in a New Family with TECTA Mutation Markus H F Pfister MD PhD (presenter); Haluk Ozkarakas MD; Guy Van Camp; Nikolaus Blin PhD; Hans Peter Zenner MD PhD; Peter Nuernberg PhD; Susan Kupka PhD Tubingen Germany; Izmit Turkey; Antwerp Belgium; Tubingen Germany; Tubingen Germany; Berlin Germany; Tubingen Germany
Problem: Alpha-tectorin is a noncollagenous component of the tectorial membrane which plays an essential role in auditory transduction. In several DFNA12 families, mutations in TECTA, the gene encoding alpha-tectorin, were shown to
POSTERS
Conclusion: Comparative genomic hybridization is suitable for screening ethmoid sinus adenocarcinoma on a genetic level. Significance: The results on these screens indicate that further genetic investigations of this rare tumor may provide more details about the tumor’s genetic abnormalities and perhaps clarify the etiology of sinonasal adenocarcinomas and its relationship with the wood dust. Support: Grant from Universidad de Oviedo AYP-02-520
Research Posters P165
Research Posters
cause hearing impairment (HI) with different phenotypes depending on the location of the DNA change. Methods: Here we report a Turkish family displaying autosomal dominant inherited HI. Linkage analysis revealed significant cosegregation of the disease to markers on chromosome 11q23.3-q24. This region contains the TECTA gene which was subsequently sequenced. Results: Clinically, the reported family shows a postlingual, progressive hearing impairment. A nucleotide change in exon 13, 4526T⬎C, was detected leading to a substitution from cysteine to glycine at codon 1509 of the TECTA protein. This cysteine is located in vWFD4 domain, which is known to be involved in disulfide bonds and protein-protein interactions. Conclusion: It is conspicuous that the phenotype in this family correlates with two other families, also displaying mutations within the vWFD domains. In all three families these mutations result in postlingual, progressive HI involving high frequencies. In contrast, mutations which are not affecting the vWFD domains seem to provoke prelingual, stable, mid-frequency sensorineural HI. Significance: This study underlines the importance of TECTA for the hearing process. Support: Else-Kro¨ ner-Fresenius-Stiftung, Dr Karl-KuhnStiftung, Flemish FWO (Fonds Wetenschappelijk onderzoek) to G.V.C.
R038 Difference of Microvessel Density and CD34 Expression between Carcinoma and Normal Mucosa Meihua Zheng MD Jinan China
Problem: Angiogenesis is an important factor for tumor growth. This study aimed to detect the microvessel density and CD34 expression in tumor tissue and in normal mucosa and to analyze the difference of microvessel density and CD34 expression between squamous cell carcinoma of head and neck and normal mucosa. Methods: There were 40 specimens from patients with squamous cell carcinoma and 9 specimens from normal mucosa. These tissuea were fixed in 4% PFA and detected according to immunhistochemical method. Results: CD34-positive expression in endothelial cells was 30 (75%) in 40 tumor specimens and 8 (89%) in 9 normal mucosa. The staining intensity of CD34 is not significant between different clinical stages, between histological grades, and between carcinoma and normal mucosa (P ⬎ 0.05). Microvessel density in tumor tissue was higher than in normal mucosa (P ⬍ 0.05). Conclusion: CD34 expression in endothelial cells can show the microvessel. In addition, microvessel density is higher in tumor tissue than in normal mucosa.
Significance: To testify the fact that angiogenesis play an important role in carcinoma growth. To restrain angiogenesis may control the growth of carcinoma. Support: CSC scholarship of China R039 Effect of IL-2 Concentration during TⴚCell Culture on Effector Phenotype and Function Michael S Srodes MD (presenter); Richard Bergstrom MD; Damon A Silverman MD; Julian A Kim MD; Hallie Graor; Jorgen Kjaergaard PhD Cleveland OH; Shaker Heights OH; Shaker Heights OH; Cleveland OH; Cleveland OH; Cleveland OH
Problem: To evaluate the effect of increasing concentrations of exogenous interleukin (IL) 2 on resultant effector T-cell phenotype and function. Methods: MCA205 fibrosarcoma was inoculated subcutaneously in mice and draining lymph nodes (LN) were harvested for ex vivo culture. Tumor-draining LN cells were activated with immobilized anti-CD3 for 48 hours and then expanded in varying concentrations of IL-2 ranging from 1 to 1000 U/mL for 72 hours. The cell cultures were compared for fold-increase in cell number, expression of activation markers, tumor-specific cytokine release in vitro, and effector function in vivo. Trafficking of activated T cells to tumor sites was evaluated by labeling with fluorescent markers and enumeration of infiltrating T cells under fluorescence microscopy. Results: A dose-dependent increase in cell numbers was observed during culture with increasing concentrations of IL-2. The percentage of cells which expressed Thy1.2, CD4, and CD8 was similar; dose-dependent changes were observed in percentage of cells upregulating CD25 and downregulating CD62L expression with increasing IL-2 concentration. Tumor-specific interferon-gamma secretion in vitro was observed from T cells cultured in 1 and 4 and but not 10, 100, or 1000 U/mL IL-2. T cells cultured in 1 and 4 U/mL IL-2 demonstrated significant therapeutic activity against 3-day established pulmonary metastases in vivo. T cells cultured in 10, 100, or 1000 U/mL IL-2 demonstrated significantly reduced therapeutic efficacy in vivo on a per-cell basis. The number of T cells migrating to the tumor site was diminished in mice that received T cells cultured with high concentrations of IL-2. Conclusion: These results suggest that the concentration of exogenous IL-2 present during T-cell culture may significantly influence effector phenotype and function. Significance: Concentrations of IL-2 which result in adequate expansion of T-cell number while preserving in vivo therapeutic efficacy should be optimized for adoptive immunotherapy protocols. Support: Resident Research Grant from the AAO-HNSF (MSS)
POSTERS
P166
Otolaryngology– Head and Neck Surgery August 2003
R035 P53 Mutations in Head and Neck Carcinoma: Correlating Current Detection Methods Thomas R Lowry MD (presenter); Joseph A Brennan MD San Antonio TX; Boerne TX
Problem: To correlate currently accepted methods of p53 mutation detection in head and neck carcinoma with quantitative rapid-cycle, real-time PCR analysis. Methods: Fresh-frozen tissue samples from 37 primary head and neck squamous cell carcinomas were subjected for analysis. Presence of p53 mutation was determined by immunohistochemical staining, genomic DNA sequencing, and quantitative rapid-cycle real-time PCR using the LightCycler. Results were correlated using standard statistical analysis. Results: 21 of 37 samples (57%) showed positive reaction for p53 protein on immunohistochemical staining. Results of genomic DNA sequencing and rapid-cycle, real time PCR using the LightCycler are presented. Conclusion: Traditional methods for detection of p53 mutations in head and neck carcinomas, including immunohistochemistry and genomic DNA sequencing, have certain limitations. p53 quantification using rapid-cycle, realtime PCR is an evolving technology which possesses advantages over these limitations. This study demonstrates that rapid-cycle, real-time PCR is an accurate and efficient method for screening head and neck carcinomas for presence of p53 mutations. Significance: Correlation of methods for p53 mutation detection in head and neck carcinomas. Support: This research is part of a larger study involving p53 analysis and real-time PCR. Total funding to date is from the Surgeon General Office in the amount of $151,000. R036 Evaluation of the Role of the Epstein-Barr Virus in 300 Primary SCCHN David Goldenberg MD (presenter); Nicole Benoit BS; William Westra MD; Wayne M Koch MD; David Sidransky MD; Joseph Califano MD Baltimore MD; Baltimore MD; Baltimore MD; Columbia MD; Baltimore MD; Baltimore MD
Problem: Recently, multiple studies have been published linking EBV with oral squamous cell carcinoma
(OSCC) and, to a lesser extent, hypopharyngeal and laryngeal tumors. These reports have varied in sample size and methodology. We used a sensitive and specific method of detection of EBV, quantitative PCR, in a large cohort of patients with head and neck squamous cell carcinoma (HNSCC) to clarify the possible role of EBV in head and neck cancers and correlate EBV status with exposure history, grade, and p53 status. Methods: We examined 300 head and neck squamous cell carcinomas (HNSCC) for the presence of EBV using quantitative PCR (QPCR) for separate detection of BAM and EBNA sequences in the EBV genome with analysis of beta-actin for quantitation of genomic DNA copy number. 84 larynx, 30 hypopharynx, 71 oropharynx, and 109 oral cavity tumors were analyzed for EBV quantity, expressed as the number of viral copies/genome/cell. Results for EBV were correlated with p53 mutation status, grade, smoking, and alcohol exposure. Results: Only 2 of 300 samples (⬍1%) were overtly positive (defined as ⬎0.1 viral copies/genome/cell). EBV DNA was detected in low levels (defined as ⬎0.01 and ⬍0.1 copies/genome/cell) in 6 of 300 tumors (2%) and in insignificant levels (defined as ⬍0.01 viral copies/genome/cell) in 68 of 300 tumors (23%). No correlation was found between overt or low-level EBV positivity and either smoking exposure, ethanol exposure, p53 status, or tumor grade. Conclusion: In the overwhelming majority of HNSC EBV does not appear to contribute to growth of a dominant clonal population with integrated EBV genome and is unlikely to be etiologic in tumor development. These very low quantities of EBV detected in a minority of HNSC may be related to the presence of EBV genome in rare lymphoid or epithelial cells adjacent to primary HNSC. Significance: This study demonstrates that although EBV presence may be found in low levels in some head and neck SCC, it is unlikely that it plays a role in tumor development. Support: None reported.
R037 Genotype-Phenotype Correlation in a New Family with TECTA Mutation Markus H F Pfister MD PhD (presenter); Haluk Ozkarakas MD; Guy Van Camp; Nikolaus Blin PhD; Hans Peter Zenner MD PhD; Peter Nuernberg PhD; Susan Kupka PhD Tubingen Germany; Izmit Turkey; Antwerp Belgium; Tubingen Germany; Tubingen Germany; Berlin Germany; Tubingen Germany
Problem: Alpha-tectorin is a noncollagenous component of the tectorial membrane which plays an essential role in auditory transduction. In several DFNA12 families, mutations in TECTA, the gene encoding alpha-tectorin, were shown to
POSTERS
Conclusion: Comparative genomic hybridization is suitable for screening ethmoid sinus adenocarcinoma on a genetic level. Significance: The results on these screens indicate that further genetic investigations of this rare tumor may provide more details about the tumor’s genetic abnormalities and perhaps clarify the etiology of sinonasal adenocarcinomas and its relationship with the wood dust. Support: Grant from Universidad de Oviedo AYP-02-520
Research Posters P165
Research Posters
cause hearing impairment (HI) with different phenotypes depending on the location of the DNA change. Methods: Here we report a Turkish family displaying autosomal dominant inherited HI. Linkage analysis revealed significant cosegregation of the disease to markers on chromosome 11q23.3-q24. This region contains the TECTA gene which was subsequently sequenced. Results: Clinically, the reported family shows a postlingual, progressive hearing impairment. A nucleotide change in exon 13, 4526T⬎C, was detected leading to a substitution from cysteine to glycine at codon 1509 of the TECTA protein. This cysteine is located in vWFD4 domain, which is known to be involved in disulfide bonds and protein-protein interactions. Conclusion: It is conspicuous that the phenotype in this family correlates with two other families, also displaying mutations within the vWFD domains. In all three families these mutations result in postlingual, progressive HI involving high frequencies. In contrast, mutations which are not affecting the vWFD domains seem to provoke prelingual, stable, mid-frequency sensorineural HI. Significance: This study underlines the importance of TECTA for the hearing process. Support: Else-Kro¨ ner-Fresenius-Stiftung, Dr Karl-KuhnStiftung, Flemish FWO (Fonds Wetenschappelijk onderzoek) to G.V.C.
R038 Difference of Microvessel Density and CD34 Expression between Carcinoma and Normal Mucosa Meihua Zheng MD Jinan China
Problem: Angiogenesis is an important factor for tumor growth. This study aimed to detect the microvessel density and CD34 expression in tumor tissue and in normal mucosa and to analyze the difference of microvessel density and CD34 expression between squamous cell carcinoma of head and neck and normal mucosa. Methods: There were 40 specimens from patients with squamous cell carcinoma and 9 specimens from normal mucosa. These tissuea were fixed in 4% PFA and detected according to immunhistochemical method. Results: CD34-positive expression in endothelial cells was 30 (75%) in 40 tumor specimens and 8 (89%) in 9 normal mucosa. The staining intensity of CD34 is not significant between different clinical stages, between histological grades, and between carcinoma and normal mucosa (P ⬎ 0.05). Microvessel density in tumor tissue was higher than in normal mucosa (P ⬍ 0.05). Conclusion: CD34 expression in endothelial cells can show the microvessel. In addition, microvessel density is higher in tumor tissue than in normal mucosa.
Significance: To testify the fact that angiogenesis play an important role in carcinoma growth. To restrain angiogenesis may control the growth of carcinoma. Support: CSC scholarship of China R039 Effect of IL-2 Concentration during TⴚCell Culture on Effector Phenotype and Function Michael S Srodes MD (presenter); Richard Bergstrom MD; Damon A Silverman MD; Julian A Kim MD; Hallie Graor; Jorgen Kjaergaard PhD Cleveland OH; Shaker Heights OH; Shaker Heights OH; Cleveland OH; Cleveland OH; Cleveland OH
Problem: To evaluate the effect of increasing concentrations of exogenous interleukin (IL) 2 on resultant effector T-cell phenotype and function. Methods: MCA205 fibrosarcoma was inoculated subcutaneously in mice and draining lymph nodes (LN) were harvested for ex vivo culture. Tumor-draining LN cells were activated with immobilized anti-CD3 for 48 hours and then expanded in varying concentrations of IL-2 ranging from 1 to 1000 U/mL for 72 hours. The cell cultures were compared for fold-increase in cell number, expression of activation markers, tumor-specific cytokine release in vitro, and effector function in vivo. Trafficking of activated T cells to tumor sites was evaluated by labeling with fluorescent markers and enumeration of infiltrating T cells under fluorescence microscopy. Results: A dose-dependent increase in cell numbers was observed during culture with increasing concentrations of IL-2. The percentage of cells which expressed Thy1.2, CD4, and CD8 was similar; dose-dependent changes were observed in percentage of cells upregulating CD25 and downregulating CD62L expression with increasing IL-2 concentration. Tumor-specific interferon-gamma secretion in vitro was observed from T cells cultured in 1 and 4 and but not 10, 100, or 1000 U/mL IL-2. T cells cultured in 1 and 4 U/mL IL-2 demonstrated significant therapeutic activity against 3-day established pulmonary metastases in vivo. T cells cultured in 10, 100, or 1000 U/mL IL-2 demonstrated significantly reduced therapeutic efficacy in vivo on a per-cell basis. The number of T cells migrating to the tumor site was diminished in mice that received T cells cultured with high concentrations of IL-2. Conclusion: These results suggest that the concentration of exogenous IL-2 present during T-cell culture may significantly influence effector phenotype and function. Significance: Concentrations of IL-2 which result in adequate expansion of T-cell number while preserving in vivo therapeutic efficacy should be optimized for adoptive immunotherapy protocols. Support: Resident Research Grant from the AAO-HNSF (MSS)
POSTERS
P166
Otolaryngology– Head and Neck Surgery August 2003