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EMS1 gene amplification correlates with poor prognosis in HNSCC
P86
Otolaryngology. Head and NeckSurgery August T999
Research Forum--Monday
Conclusion: FISH is an ideal technique for analysis of chromosomal aberrations in paraffin-embedded, archival tissue specimens. While chromosome 20Q amplification has been suggested as a possible mediator of tumorigenesis in head and neck cancers, using FISH, we have shown no direct evidence of either amplification or deletion of the specific region 20q13 in head and neck squamous carcinoma. Poster 25
Expression of FGF-BP in Squarnous Cancer of the Head and Neck WEIMIN LI MD PHD (presenter); JEZHEN LIN MD; STEVENK JUHN MD; FRANK G ONDREY MD PHD; GEORGE L ADAMS MD; Minneapolis MN
Problem: The growth and metastatic spread of head and neck tumors is directly related to tumor angiogenesis, which is controlled by a variety of mediators. Earlier studies showed that a secreted binding protein for fibroblast growth factors (FGF-BP) is expressed at high levels in squamous cell carcinoma (SCC) cell lines. Overexpression studies or conversely reduced expressions of FGF-BP by ribozyme targeting have indicated a direct role of this protein in angiogenesis during tumor development, We designed this pilot study to characterize FGF-BP expression in head and neck tumor specimens using RT-PCR and sequencing. Methods: Tissues used for this study were obtained from 3 specimens from oral and larynx cancer patients at our university hospital. Total cellular RNA was extracted with guanidium isocyanate acidic phenol chloroform. The expression of FGF-BP mRNA was performed by RT-PCR using primer sequences from published cDNA sequences (Genebank). The cDNAs were cloned into pBluscript II vector. E coli was transformed with plasmid DNA using the heat-shock method. The amplified plasmid DNA was purified and automatically sequenced. Sequences were confirmed with published sequences using BLAST 2.0.6 software. Results: Overlapping cDNA clones encoding squamous cell carcinoma FGF-BP were isolated by RT-PCR. Three plasmid constructs containing ligated PCR amplification products for each tumor specimen were generated and found to be of the predicted size. The clones used were pBluscript FGF-BP containing 702 bp insert of human FGF-BP cDNA. Sequence analysis demonstrated greater than 99% homology with the published cDNA sequences for each specimen. Conclusion: The results of this pilot study demonstrated that FGF-BP is present in squamous cell carcinomas of the head and neck, leading us to conclude that FGF-BP may be an angiogenic factor that plays a significant role in tumor growth and metastasis in HNSCC. Clinical Significance: We hope that this study will eventually provide a marker for the growth and metastatic potential of squamous cell carcinoma of the head and neck. (Supported by Lions Research Foundation.)
Poster 26
Blocking TGF-beta Signaling Accelerates $quamous Cell Carcinogenesis CYNTHIA GO MD PHD (presenter); XIAO-JING WANG MD PHD; Houston TX
Problem: The transforming growth factor-~ (TGFB) family has multiple functions in regulating cell proliferation and differentiation. The interaction between TGFB and its receptor (TGFBR) is critical to the growth-inhibitory effects seen in epithelial cells. Mutations in the TGFBR have been shown to be present in squamous cell cancer of the head and neck. This study was undertaken to determine the causal r01eof the loss of the TGFBR in Cancer development. Methods: Transgenic mice were created using a construct that rendered the TGFBR inactive in the epidermis. A truncated mouse loricrin promoter combined with a modified inactive TGFBR gene allowed expression of the modified TGFBR in epidermal cells only. These mice were subjected to a 2-stage chemical carcinogenesis protocol. Skin tumors from these mice were examined by flow cytometry to determine their cell cycle status. In addition, gene expression of inhibitors of cell cycle progression as well as angiogenesis factors were tested. Results: TGFBR transgenic mice exhibited thickened skin, resulting from epidermal hyperproliferation. After carcinogenic stimulus, these mice showed increased sensitivity, with earlier appearance and increased number of squamous papillomas, and an increase in progression to squamous cell carcinomas. Tumor cell flow cytometry showed reduction in the G 1 and increase in the G2/M population. These tumors also exhibited loss of expression of p21 and p15, inhibitors of cyclin-dependent kinases. Furthermore, there was an increase in expression of vascular endothelial growth factor (an angiogenesis factor) and a decrease in expression of thrombospondin-1 (an angiogenesis inhibitor). Conclusion: These data provide in vivo evidence that inactivation of the TGFB/TGFBR pathway accelerates skin tumorigenesis. The mechanism by which this occurs is by a loss of cell cycle control and by an increase in tumor angiogenesis. Clinical Significance: Since the majority of tumors of the head and neck are squamous cell carcinomas, an understanding of the mechanisms by which carcinogenesis occurs in epithelial cells will allow for the molecular identification of more aggressive tumors (ie, those with mutations in the TGFBR) and provide biochemotherapeutic options for treatment of these cancers (ie, gene therapy in TGFBR mutant tumors). (Supported by a Career Development Award [to X-J. W.] from the Dermatology Foundation.) Poster 27
EM$1 Gene Amplification Correlates with Poor Prognosis in HNSCC JUAN P RODRIGO MD; LUIS A GARCIA MD; PEDRO S LAZO PHD; SOFIA RAMOS PHD; CARLOS SUAREZ MD (presenter); Oviedo Asturias Spain
OtolaryngologyHead and Neck Surgery
Research Forum--Monday
Volume 121 Number 2
Problem: Chromosome l lq13 amplification has been found frequently in head and neck squamous cell carcinomas (HNSCCs), indicating poor prognosis. The EMS1 gene has been identified as one of the "active" oncogenes in this region because it is overexpressed in all carcinomas with EMS1 amplification, and this overexpression causes increased cell motility and invasion in vitro. However, little is known about the prognostic significance of individual EMS 1 amplification in HNSCC. Our objective was to further address the frequency of EMS 1 amplification in HNSCC and to evaluate its correlation with clinicopathologic variables, relapse, and survival. Methods: Tissue samples from 104 consecutive patients with primary HNSCC who underwent surgical resection of their tumors were studied. After DNA extraction, the EMS 1 copy number in tumor samples was estimated with the polymerase chain reaction method. The presence or absence of amplification was correlated with the anatomic site, T stage, nodal involvement, pathologic grade, disease recurrence, distant metastasis, clinical outcome (disease status), and survival. Patients were observed for at least 36 months (mean followup 54 months). Results: EMS1 amplification was identified in 21 cases (20%). Amplification was related with advanced T stages (P = 0.003), lymph node involvement (P = 0.02), and poor histologic differentiation (P = 0.018). Recurrent disease was identiffed in 76% (16/21) of cases with amplification and 39% (32/83) of cases without amplification (P = 0.006). Seventeen patients (81%) with EMS1 amplification and 35 patients (42%) without amplification died of disease (P = 0.003). EMS1 amplification status, individually, had a significant effect on survival (the 3-year survival was 54% in nonamplifled cases and 7% in amplified cases, P < 0.0001). In multivariate analysis (Cox regression model), EMS1 amplification was found to be an independent predictor of mortality by the tumor (P = 0.003). Conclusion: Amplification of the EMS1 gene seems to be an important biological marker indicating poor prognosis in HNSCC, independent of clinical stage, histologic differentiation, or anatomic site. This may confirm its role as an "active" oncogene in the llq13 region. Poster 28
Expression of E-selectin Ligand in Primary Head and Neck Squamous Cell Carcinoma YOUNG J KIM MD PHD (presenter); ROBERT A WEISMAN MD FACS; AJIT VARKI MD; NISSI VARKI MD; HULLING HAN MD; JOANNE YI MD; La Jolla CA; San Diego CA; La Jolla CA; La Jolla CA; La Jolla CA; San Diego CA
Problem." Sialyl Lewis X (SLe-X) is a tetrasaccharide structure expressed on various carcinomas, and it is implicated as one of the adhesion molecules responsible for the interactions between cancer cells and endothelium, a step that is
P87
thought to be important in the evolution of hematogenous metastasis. SLe-X is an element of E-selectin ligand (ESL), but not the ligand itself. SLe-X must be presented on a macromolecular glycoprotein in order to be recognized by Eselectin. Carcinoma cells may express SLe-X structures, but they may not necessarily express ESL. It has been reported that there is enhanced expression of SLe-X structures in metastatic squamous cell cancer of the head and neck relative to the primary site, but there is no correlation between ESL and disease status. Methods: Ten paraffin-embedded primary tumor specimens were stained with soluble human E-selectin with an enterokinase substrate epitope tag. After deparaffination, formalin fixation, and blocking, the tissues were incubated with E-selectin complexed with mouse anti-FLAG antibody (against the enterokinase substrate). Detection of mouse antibody was screened with anti-mouse antibody conjugated to alkaline phosphatase and subsequently stained for phosphatase activity. Results: Seven of the 10 specimens stained positively with ESL expression. E-selectin binding to its ligand is calciumdependent, and the presence of EDTA abrogated the staining. It is concluded that ESL is frequently expressed on head and neck squamous cell primary cancers. Clinical Significance: Because ESL is more fundamentally involved in potential cancer cell/endothelial cell interactions than SLe-X, it may prove to be a better marker to study metastatic behavior in head and neck squamous cell cancer. Poster 29
MUC4 (Sialomucin Complex) Expression in Salivary Tumors and SCC of the Upper Aerodigestive Tract DONALD T WEED MD (presenter); KERMIT CARRAWAY PHD; MARIA CARVAJAL MBA; THOMAS LEE MD; JEFFREY PACHECO MD; CARMEN GOMEZ-FERNANDEZ MD; ADAM BELLO MD; W JARRARD GOODWIN MD; Miami FL
Objective: Peptide sequence homology between the gene product of human MUC4 and rat sialomucin complex (SMC) has recently been reported. Each contains a mucin subunit with antiadhesive activity linked to the plasma membrane via a transmembrane subunit with 2 epidermal growth factor-like domains that in the rat act as ligands for ErbB2. Altered expression of MUC4 may lead to transformation to a neoplastic phenotype and aid in the development of metastatic potential of the transformed cell. This study investigates normal and neoplastic MUC4 expression in squamous cell carcinoma (SCC) of the upper aerodigestive tract (UADT) and in salivary neoplasms. Methods: Localization of MUC4 in tumor and adjacent normal tissue is determined by immunocytochemical studies using antibodies prepared against both subunits of the rat SMC. Fresh-frozen tissues from surgical resection specimens are further analyzed by Western blot. Specimens are solubi-
Otolaryngology. Head and NeckSurgery August T999
Research Forum--Monday
Conclusion: FISH is an ideal technique for analysis of chromosomal aberrations in paraffin-embedded, archival tissue specimens. While chromosome 20Q amplification has been suggested as a possible mediator of tumorigenesis in head and neck cancers, using FISH, we have shown no direct evidence of either amplification or deletion of the specific region 20q13 in head and neck squamous carcinoma. Poster 25
Expression of FGF-BP in Squarnous Cancer of the Head and Neck WEIMIN LI MD PHD (presenter); JEZHEN LIN MD; STEVENK JUHN MD; FRANK G ONDREY MD PHD; GEORGE L ADAMS MD; Minneapolis MN
Problem: The growth and metastatic spread of head and neck tumors is directly related to tumor angiogenesis, which is controlled by a variety of mediators. Earlier studies showed that a secreted binding protein for fibroblast growth factors (FGF-BP) is expressed at high levels in squamous cell carcinoma (SCC) cell lines. Overexpression studies or conversely reduced expressions of FGF-BP by ribozyme targeting have indicated a direct role of this protein in angiogenesis during tumor development, We designed this pilot study to characterize FGF-BP expression in head and neck tumor specimens using RT-PCR and sequencing. Methods: Tissues used for this study were obtained from 3 specimens from oral and larynx cancer patients at our university hospital. Total cellular RNA was extracted with guanidium isocyanate acidic phenol chloroform. The expression of FGF-BP mRNA was performed by RT-PCR using primer sequences from published cDNA sequences (Genebank). The cDNAs were cloned into pBluscript II vector. E coli was transformed with plasmid DNA using the heat-shock method. The amplified plasmid DNA was purified and automatically sequenced. Sequences were confirmed with published sequences using BLAST 2.0.6 software. Results: Overlapping cDNA clones encoding squamous cell carcinoma FGF-BP were isolated by RT-PCR. Three plasmid constructs containing ligated PCR amplification products for each tumor specimen were generated and found to be of the predicted size. The clones used were pBluscript FGF-BP containing 702 bp insert of human FGF-BP cDNA. Sequence analysis demonstrated greater than 99% homology with the published cDNA sequences for each specimen. Conclusion: The results of this pilot study demonstrated that FGF-BP is present in squamous cell carcinomas of the head and neck, leading us to conclude that FGF-BP may be an angiogenic factor that plays a significant role in tumor growth and metastasis in HNSCC. Clinical Significance: We hope that this study will eventually provide a marker for the growth and metastatic potential of squamous cell carcinoma of the head and neck. (Supported by Lions Research Foundation.)
Poster 26
Blocking TGF-beta Signaling Accelerates $quamous Cell Carcinogenesis CYNTHIA GO MD PHD (presenter); XIAO-JING WANG MD PHD; Houston TX
Problem: The transforming growth factor-~ (TGFB) family has multiple functions in regulating cell proliferation and differentiation. The interaction between TGFB and its receptor (TGFBR) is critical to the growth-inhibitory effects seen in epithelial cells. Mutations in the TGFBR have been shown to be present in squamous cell cancer of the head and neck. This study was undertaken to determine the causal r01eof the loss of the TGFBR in Cancer development. Methods: Transgenic mice were created using a construct that rendered the TGFBR inactive in the epidermis. A truncated mouse loricrin promoter combined with a modified inactive TGFBR gene allowed expression of the modified TGFBR in epidermal cells only. These mice were subjected to a 2-stage chemical carcinogenesis protocol. Skin tumors from these mice were examined by flow cytometry to determine their cell cycle status. In addition, gene expression of inhibitors of cell cycle progression as well as angiogenesis factors were tested. Results: TGFBR transgenic mice exhibited thickened skin, resulting from epidermal hyperproliferation. After carcinogenic stimulus, these mice showed increased sensitivity, with earlier appearance and increased number of squamous papillomas, and an increase in progression to squamous cell carcinomas. Tumor cell flow cytometry showed reduction in the G 1 and increase in the G2/M population. These tumors also exhibited loss of expression of p21 and p15, inhibitors of cyclin-dependent kinases. Furthermore, there was an increase in expression of vascular endothelial growth factor (an angiogenesis factor) and a decrease in expression of thrombospondin-1 (an angiogenesis inhibitor). Conclusion: These data provide in vivo evidence that inactivation of the TGFB/TGFBR pathway accelerates skin tumorigenesis. The mechanism by which this occurs is by a loss of cell cycle control and by an increase in tumor angiogenesis. Clinical Significance: Since the majority of tumors of the head and neck are squamous cell carcinomas, an understanding of the mechanisms by which carcinogenesis occurs in epithelial cells will allow for the molecular identification of more aggressive tumors (ie, those with mutations in the TGFBR) and provide biochemotherapeutic options for treatment of these cancers (ie, gene therapy in TGFBR mutant tumors). (Supported by a Career Development Award [to X-J. W.] from the Dermatology Foundation.) Poster 27
EM$1 Gene Amplification Correlates with Poor Prognosis in HNSCC JUAN P RODRIGO MD; LUIS A GARCIA MD; PEDRO S LAZO PHD; SOFIA RAMOS PHD; CARLOS SUAREZ MD (presenter); Oviedo Asturias Spain
OtolaryngologyHead and Neck Surgery
Research Forum--Monday
Volume 121 Number 2
Problem: Chromosome l lq13 amplification has been found frequently in head and neck squamous cell carcinomas (HNSCCs), indicating poor prognosis. The EMS1 gene has been identified as one of the "active" oncogenes in this region because it is overexpressed in all carcinomas with EMS1 amplification, and this overexpression causes increased cell motility and invasion in vitro. However, little is known about the prognostic significance of individual EMS 1 amplification in HNSCC. Our objective was to further address the frequency of EMS 1 amplification in HNSCC and to evaluate its correlation with clinicopathologic variables, relapse, and survival. Methods: Tissue samples from 104 consecutive patients with primary HNSCC who underwent surgical resection of their tumors were studied. After DNA extraction, the EMS 1 copy number in tumor samples was estimated with the polymerase chain reaction method. The presence or absence of amplification was correlated with the anatomic site, T stage, nodal involvement, pathologic grade, disease recurrence, distant metastasis, clinical outcome (disease status), and survival. Patients were observed for at least 36 months (mean followup 54 months). Results: EMS1 amplification was identified in 21 cases (20%). Amplification was related with advanced T stages (P = 0.003), lymph node involvement (P = 0.02), and poor histologic differentiation (P = 0.018). Recurrent disease was identiffed in 76% (16/21) of cases with amplification and 39% (32/83) of cases without amplification (P = 0.006). Seventeen patients (81%) with EMS1 amplification and 35 patients (42%) without amplification died of disease (P = 0.003). EMS1 amplification status, individually, had a significant effect on survival (the 3-year survival was 54% in nonamplifled cases and 7% in amplified cases, P < 0.0001). In multivariate analysis (Cox regression model), EMS1 amplification was found to be an independent predictor of mortality by the tumor (P = 0.003). Conclusion: Amplification of the EMS1 gene seems to be an important biological marker indicating poor prognosis in HNSCC, independent of clinical stage, histologic differentiation, or anatomic site. This may confirm its role as an "active" oncogene in the llq13 region. Poster 28
Expression of E-selectin Ligand in Primary Head and Neck Squamous Cell Carcinoma YOUNG J KIM MD PHD (presenter); ROBERT A WEISMAN MD FACS; AJIT VARKI MD; NISSI VARKI MD; HULLING HAN MD; JOANNE YI MD; La Jolla CA; San Diego CA; La Jolla CA; La Jolla CA; La Jolla CA; San Diego CA
Problem." Sialyl Lewis X (SLe-X) is a tetrasaccharide structure expressed on various carcinomas, and it is implicated as one of the adhesion molecules responsible for the interactions between cancer cells and endothelium, a step that is
P87
thought to be important in the evolution of hematogenous metastasis. SLe-X is an element of E-selectin ligand (ESL), but not the ligand itself. SLe-X must be presented on a macromolecular glycoprotein in order to be recognized by Eselectin. Carcinoma cells may express SLe-X structures, but they may not necessarily express ESL. It has been reported that there is enhanced expression of SLe-X structures in metastatic squamous cell cancer of the head and neck relative to the primary site, but there is no correlation between ESL and disease status. Methods: Ten paraffin-embedded primary tumor specimens were stained with soluble human E-selectin with an enterokinase substrate epitope tag. After deparaffination, formalin fixation, and blocking, the tissues were incubated with E-selectin complexed with mouse anti-FLAG antibody (against the enterokinase substrate). Detection of mouse antibody was screened with anti-mouse antibody conjugated to alkaline phosphatase and subsequently stained for phosphatase activity. Results: Seven of the 10 specimens stained positively with ESL expression. E-selectin binding to its ligand is calciumdependent, and the presence of EDTA abrogated the staining. It is concluded that ESL is frequently expressed on head and neck squamous cell primary cancers. Clinical Significance: Because ESL is more fundamentally involved in potential cancer cell/endothelial cell interactions than SLe-X, it may prove to be a better marker to study metastatic behavior in head and neck squamous cell cancer. Poster 29
MUC4 (Sialomucin Complex) Expression in Salivary Tumors and SCC of the Upper Aerodigestive Tract DONALD T WEED MD (presenter); KERMIT CARRAWAY PHD; MARIA CARVAJAL MBA; THOMAS LEE MD; JEFFREY PACHECO MD; CARMEN GOMEZ-FERNANDEZ MD; ADAM BELLO MD; W JARRARD GOODWIN MD; Miami FL
Objective: Peptide sequence homology between the gene product of human MUC4 and rat sialomucin complex (SMC) has recently been reported. Each contains a mucin subunit with antiadhesive activity linked to the plasma membrane via a transmembrane subunit with 2 epidermal growth factor-like domains that in the rat act as ligands for ErbB2. Altered expression of MUC4 may lead to transformation to a neoplastic phenotype and aid in the development of metastatic potential of the transformed cell. This study investigates normal and neoplastic MUC4 expression in squamous cell carcinoma (SCC) of the upper aerodigestive tract (UADT) and in salivary neoplasms. Methods: Localization of MUC4 in tumor and adjacent normal tissue is determined by immunocytochemical studies using antibodies prepared against both subunits of the rat SMC. Fresh-frozen tissues from surgical resection specimens are further analyzed by Western blot. Specimens are solubi-