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Expression of very late antigen (β1) integrins in pleomorphic adenoma cell lines
Otolaryngology Head and Neck Surgery P 84
Scientific Sessions-- Monday
ations in pleomorphic adenomas at microsatellite loci in regions reported to be sites of cytogenetic abnormalities. A polymerase chain reaction-based single-strand conformation polymorphism analysis was used to identify loss of heterozygosity (LOH) and microsatellite instability in pleomorphic adenoma specimens. DNA extracted from 36 freshfrozen paired normal and tumor specimens from 18 patients (18 normal, 14 pleomorphic adenoma, and four pleomorphic adenoma with a foci of carcinoma ex pleomorphic adenoma) was analyzed for microsatellite alterations at loci on chromosomes 3p, 6q, 8p, and 8q. Correlation with clinical and pathologic features was performed. Overall, 7 of 18 cases (39%) manifested LOH at the loci tested. LOH was noted on 3p, 6q, 8p, and 8q microsatellite loci in 5.6%, 13.0%, 15%, and 27% of informative cases, respectively. No microsatellite instability was noted at the loci analyzed. Specimens from patients with carcinoma e x pleomorphic adenoma did not manifest enhanced LOH. We observed a lack of association between chromosomal abnormalities and age, sex, tumor size, histologic features, and DNA ploidy in these tumors. A subset of pleomorphic adenomas exhibited loss of heterozygosity at microsatellite loci on 3p, 6q, 8p, and 8q. The incidence did not appear to be increased in patients with focal carcinoma ex pleomorphic adenoma, indicating that LOH at these loci is an early event in tumorigenesis. In addition, our results indicate that genetic alterations other than translocations are involved in the genesis of these neoplasms. Further evaluation at these loci is needed to identify potential tumor suppressor genes that function in initiation and progression of pleomorphic adenomas. Poster 30
Expression of Very Late Antigen (I~) Integrins in Pleomorphic Adenoma Cell Lines MELISSA G. STEINER,PHD (presenter), WILLIAM I. KUHEL, MD, JOHN F. CAREW, MD, and JERRY HUO, MD, New York, N.Y.
Pleomorphic adenomas are benign salivary neoplasms that are prone to recur if their capsule is violated or if they are inadequately excised. Cell surface molecules that function in cell-cell and cell-extracellular matrix interactions may play a role in their pathophysiology. Integrins are a group of related transmembrane, heterodimer glycoproteins that function in cell-cell and cell-extracellular matrix interactions. They consist of noncovalently associated ~ and 13 subunits and are grouped into families based on their ~ subunit. The very late antigen (VLA) integrins share a common [3 subunit (~1) and recognize specific extracellular matrix components including collagen, fibronectin, and laminin, depending on their ~ subunit. This study reports the expression of VLA (130 integrins in pleomorphic adenoma cell lines. Specimens of pleomorphic adenomas from patients undergoing parotidectomy were obtained and propagated in monolayer tissue cultures. Monoclonal antibodies directed against the subunits of the VLA integrin family (r162 ~v, and ~1) were used for indirect immunofluorescence staining
August 1995
(six cell lines) and flow cytometric analysis (five cell lines). The cell lines from the pleomorphic adenomas demonstrated epithelial morphology and positive immunohistochemical staining for cytokeratin-14 and ~-actin, which is characteristic of myoepithelial cells. Immunofluorescence staining revealed particularly strong expression of the ~3, c~v and 131subunits. The ~:, ~4, and o~5 subunits showed weaker immunofluorescence staining. Flow cytometric evaluation revealed the same general pattern of integrin expression. There was a fivefold increase in mean fluorescence (relative to isotype control) for all cell lines with respect to ~ and 131 integrin subunits. Four of five lines demonstrated a fivefold increase in expression of the o~3 subunit. To confirm the validity of this in vitro model for the study of integrins in pleomorphic adenomas, these results must be compared with integrin expression in fresh pleomorphic adenoma tissue samples. Poster 31
Expression of Very Late Antigen (15I) Integrinsin Pleomorphic Adenomas MELISSA G. STEINER,PHD (presenter), WILLIAM I. KUHEL, MD, JOHN F. CAREW, MD, and JERRY HUO, MD, New York, N.Y.
Integrins are a group of related transmembrane, heterodimer glycoproteins that function in cell-cell and cellextracellular matrix interactions. They are composed of noncovalently associated ~ and 13 subunits and are grouped into families based on their 13subunit. The very late antigen (VLA) family of integrins share a common 131 subunit. Depending on the associated ~ subunit, integrins of the VLA family interact with specific extracetlular matrix components, including collagen, fibronectin, and laminin. The VLA integrins may be involved in the cell-extracellular matrix interactions that enable neoplastic cells from pleomorphic adenomas to implant in dermal and subcutaneous tissues when the tumor capsule is inadvertently ruptured. This study investigated the expression of the VLA ([3~) integrin family in six pleomorphic adenomas and five specimens of normal parotid tissue using immunohistochemical techniques. Specimens of pleomorphic adenomas from patients undergoing parotidectomy were obtained. Portions of tumor and grossly normal parotid tissue distant from the tumor were obtained and cryopreserved for later analysis. Standard immunohistochemistry was performed on 5 gm cryosections using monoclonal antibodies directed against the subunits of the 131 integrin family (0~2_5, ~v and 13~). Immunoperoxidase analysis of the cryosections revealed that all normal and neoplastic parotid specimens stained positively for the integrins analyzed. Expression of ~t2, ot4, and ocv in normal tissue was confined to ductal elements; however, the expression pattern of these integrins in the tumors was more diffuse. The pattern of integrin expression in pleomorphic adenomas of the parotid may support a specific role for adhesion molecules in the cell-extracellular matrix interactions involved in tumor recurrence.
Scientific Sessions-- Monday
ations in pleomorphic adenomas at microsatellite loci in regions reported to be sites of cytogenetic abnormalities. A polymerase chain reaction-based single-strand conformation polymorphism analysis was used to identify loss of heterozygosity (LOH) and microsatellite instability in pleomorphic adenoma specimens. DNA extracted from 36 freshfrozen paired normal and tumor specimens from 18 patients (18 normal, 14 pleomorphic adenoma, and four pleomorphic adenoma with a foci of carcinoma ex pleomorphic adenoma) was analyzed for microsatellite alterations at loci on chromosomes 3p, 6q, 8p, and 8q. Correlation with clinical and pathologic features was performed. Overall, 7 of 18 cases (39%) manifested LOH at the loci tested. LOH was noted on 3p, 6q, 8p, and 8q microsatellite loci in 5.6%, 13.0%, 15%, and 27% of informative cases, respectively. No microsatellite instability was noted at the loci analyzed. Specimens from patients with carcinoma e x pleomorphic adenoma did not manifest enhanced LOH. We observed a lack of association between chromosomal abnormalities and age, sex, tumor size, histologic features, and DNA ploidy in these tumors. A subset of pleomorphic adenomas exhibited loss of heterozygosity at microsatellite loci on 3p, 6q, 8p, and 8q. The incidence did not appear to be increased in patients with focal carcinoma ex pleomorphic adenoma, indicating that LOH at these loci is an early event in tumorigenesis. In addition, our results indicate that genetic alterations other than translocations are involved in the genesis of these neoplasms. Further evaluation at these loci is needed to identify potential tumor suppressor genes that function in initiation and progression of pleomorphic adenomas. Poster 30
Expression of Very Late Antigen (I~) Integrins in Pleomorphic Adenoma Cell Lines MELISSA G. STEINER,PHD (presenter), WILLIAM I. KUHEL, MD, JOHN F. CAREW, MD, and JERRY HUO, MD, New York, N.Y.
Pleomorphic adenomas are benign salivary neoplasms that are prone to recur if their capsule is violated or if they are inadequately excised. Cell surface molecules that function in cell-cell and cell-extracellular matrix interactions may play a role in their pathophysiology. Integrins are a group of related transmembrane, heterodimer glycoproteins that function in cell-cell and cell-extracellular matrix interactions. They consist of noncovalently associated ~ and 13 subunits and are grouped into families based on their ~ subunit. The very late antigen (VLA) integrins share a common [3 subunit (~1) and recognize specific extracellular matrix components including collagen, fibronectin, and laminin, depending on their ~ subunit. This study reports the expression of VLA (130 integrins in pleomorphic adenoma cell lines. Specimens of pleomorphic adenomas from patients undergoing parotidectomy were obtained and propagated in monolayer tissue cultures. Monoclonal antibodies directed against the subunits of the VLA integrin family (r162 ~v, and ~1) were used for indirect immunofluorescence staining
August 1995
(six cell lines) and flow cytometric analysis (five cell lines). The cell lines from the pleomorphic adenomas demonstrated epithelial morphology and positive immunohistochemical staining for cytokeratin-14 and ~-actin, which is characteristic of myoepithelial cells. Immunofluorescence staining revealed particularly strong expression of the ~3, c~v and 131subunits. The ~:, ~4, and o~5 subunits showed weaker immunofluorescence staining. Flow cytometric evaluation revealed the same general pattern of integrin expression. There was a fivefold increase in mean fluorescence (relative to isotype control) for all cell lines with respect to ~ and 131 integrin subunits. Four of five lines demonstrated a fivefold increase in expression of the o~3 subunit. To confirm the validity of this in vitro model for the study of integrins in pleomorphic adenomas, these results must be compared with integrin expression in fresh pleomorphic adenoma tissue samples. Poster 31
Expression of Very Late Antigen (15I) Integrinsin Pleomorphic Adenomas MELISSA G. STEINER,PHD (presenter), WILLIAM I. KUHEL, MD, JOHN F. CAREW, MD, and JERRY HUO, MD, New York, N.Y.
Integrins are a group of related transmembrane, heterodimer glycoproteins that function in cell-cell and cellextracellular matrix interactions. They are composed of noncovalently associated ~ and 13 subunits and are grouped into families based on their 13subunit. The very late antigen (VLA) family of integrins share a common 131 subunit. Depending on the associated ~ subunit, integrins of the VLA family interact with specific extracetlular matrix components, including collagen, fibronectin, and laminin. The VLA integrins may be involved in the cell-extracellular matrix interactions that enable neoplastic cells from pleomorphic adenomas to implant in dermal and subcutaneous tissues when the tumor capsule is inadvertently ruptured. This study investigated the expression of the VLA ([3~) integrin family in six pleomorphic adenomas and five specimens of normal parotid tissue using immunohistochemical techniques. Specimens of pleomorphic adenomas from patients undergoing parotidectomy were obtained. Portions of tumor and grossly normal parotid tissue distant from the tumor were obtained and cryopreserved for later analysis. Standard immunohistochemistry was performed on 5 gm cryosections using monoclonal antibodies directed against the subunits of the 131 integrin family (0~2_5, ~v and 13~). Immunoperoxidase analysis of the cryosections revealed that all normal and neoplastic parotid specimens stained positively for the integrins analyzed. Expression of ~t2, ot4, and ocv in normal tissue was confined to ductal elements; however, the expression pattern of these integrins in the tumors was more diffuse. The pattern of integrin expression in pleomorphic adenomas of the parotid may support a specific role for adhesion molecules in the cell-extracellular matrix interactions involved in tumor recurrence.