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Immunolocalization of Activated Transforming Growth Factor  1 in Juvenile Nasopharyngeal Angiofibroma
David G. Dillard, MD;
Cynthia Cohen, MD;
Susan Muller, DMD, MS;
John Del Gaudio, MD;
Owen Reichman, MD;
Beryl Parrish, MD;
David Rackley, BS;
Anthony A. Gal, MD
Arch Otolaryngol Head Neck Surg. 2000;126:723-725.
ABSTRACT
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Background Juvenile nasopharyngeal angiofibroma (JNA) is a histologically benign, locally aggressive neoplasm of the nasopharynx that exclusively affects male adolescents. It is known to be sensitive to androgens, but there are likely intermediary cytokines and/or growth factors that mediate aggressive stromal cell proliferation and angiogenesis. Transforming growth factor  1 (TGF-1) is a polypeptide that is secreted in an inactive form, cleaved to produce an active form, and then deactivated in the tissues. It activates fibroblast proliferation and is known to induce angiogenesis.
Objectives To evaluate the presence of activated TGF-1 within the stroma of JNA specimens and to quantify the percentage of JNA specimens expressing the active growth factor.
Design Immunohistochemical analysis was performed on 19 specimens of JNA using a unique antibody that identifies only the activated form of TGF-1. The percentage of cells staining positively for activated TGF-1 was determined semiquantitatively by visual methods.
Results Of 19 cases stained, all 19 (100%) showed strong positive staining (2 cases with 33%-66% of cells staining and 17 with 66%-100% of cells staining). Activated TGF-1 was identified in stromal cell nuclei and cytoplasm and in the endothelium of the capillaries within all specimens of JNA.
Conclusions The localization of activated TGF-1 to the fibroblasts and endothelial cells within JNA tumors suggests that TGF-1 may play a role in the stromal cell proliferation and angiogenesis associated with JNA. Additional receptor studies and more quantitative methods of analysis are needed to further define the role of TGF-1 in the pathogenesis of JNA.
INTRODUCTION
JUVENILE NASOPHARYNGEAL angiofibroma (JNA) is a histologically benign yet locally aggressive vascular head and neck neoplasm. It is an uncommon tumor that affects male adolescents almost exclusively, with a reported incidence of between 1 in 6000 and 1 in 60,000 otolaryngology patients and accounts for 0.5% of all head and neck neoplasms.1-4 Evidence of intracranial spread occurs in 10% to 20% of cases. The average age at onset of symptoms is 15 years.5
The pathogenesis of JNA is unclear. Suggested theories include androgens acting on embryonal cartilage, a hamartomatous nidus of inferior turbinate, or normal nasopharyngeal fibrovascular stroma located in the nasopharynx.1-4 The interrelationship between steroid sex hormones and the stromal and vascular proliferation may involve angiogenic growth factors and unknown cytokines. Very few studies have explored the role of various growth factors in JNA.
Transforming growth factor 1 (TGF-1) is a polypeptide growth factor associated with fibroblast proliferation and angiogenesis. It is secreted in an inactive form, which is then proteolytically cleaved, resulting in a short-lived activated form.6-7 To potentially define the role of TGF-1 in the pathogenesis of JNA, we immunohistochemically evaluated 19 cases of JNA for the activated form of TGF-1.
MATERIALS AND METHODS
Cases of JNA were reviewed from the surgical pathology files of Emory University Hospital, Atlanta, Ga, for the period 1985 to 1998. Nineteen acceptable cases of JNA were identified. The hematoxylin-eosin–stained slides from each case were reviewed by 2 pathologists (S.M. and A.A.G.) to confirm the diagnosis. Five-micrometer-thick sections of paraffin-embedded tissue blocks were processed for immunohistochemical analysis with an avidin-biotin complex kit (LSAB 2 HRP; Dako Corp, Carpinteria, Calif) and steam antigen retrieval (Autostainer; Dako Corp). The primary antibody, a polyclonal chicken anti–human antibody (R & D Systems, Minneapolis, Minn) specific for the activated form of TGF-1, was used at a dilution of 1:40. The antibody is affinity purified, and its specificity was confirmed by Western blot technique using recombinant TGF-1 according to the package insert. The secondary antibody, a rabbit anti–chicken antiserum (Chemicon International Inc, Temecula, Calif) was used at a dilution of 1:80. Positive controls consisted of human myometrial blood vessels in tissue sections. Human myometrium was chosen for a positive control because of its large content of arterioles, which are known to stain positively for TGF-1. For negative control, the primary antibody was replaced by buffer.
Sections were deparaffinized and rehydrated, then steamed in citrate buffer (pH, 6) for 20 minutes and cooled for 5 minutes before immunostaining. All tissues were then exposed to 3% hydrogen peroxide for 5 minutes, primary antibody for 25 minutes, biotinylated secondary linking antibody for 25 minutes, avidin-biotinylate enzyme complex for 25 minutes, diaminobenzidine as chromogen for 5 minutes, and hematoxylin as counterstain for 1 minute. These incubations were performed at room temperature; between incubations, sections were washed with buffer.
Immunostaining, which was nuclear and cytoplasmic in distribution, was assessed by one of us (C.C.) as 0 to 3+ intensity and semiquantitated according to the percentage of endothelial and stromal cells staining. Less than 5% of nuclear staining was regarded as negative. Also, the specimens were evaluated for the presence of normal respiratory mucosa attached to the specimen. The staining pattern of TGF-1 was evaluated in those specimens with normal respiratory mucosa in regard to endothelium, epithelium, and connective tissue staining.
Because TGF-1 is involved in scarring, it was suggested that the presence of TGF-1 might be related to preoperative embolization. Medical records and radiology archives were obtained and reviewed for the timing of embolization prior to surgical extirpation. Subsequently, these data were correlated to the specimen staining patterns.
RESULTS
All 19 cases (100%) demonstrated strong positive staining (2 cases with 33%-66% of cells staining and 17 with 66%-100% of cells staining). Activated TGF-1 was identified in both stromal cell nuclei and cytoplasm and in the endothelium of the capillaries within all specimens of JNA. The pattern of staining was consistent throughout the tumor, both in the periphery and in the central portions of the tumor. Uniformly, more than 90% of JNA stromal cells immunolocalized expression of activated TGF-1 within the tumor (Figure 1). The negative and positive controls stained appropriately.
The overlying respiratory epithelium and submucosa were normal in 14 specimens. Evaluation of the mucosa showed staining of the arteriolar endothelium but not the venular or capillary endothelium. The respiratory epithelium showed strong staining with TGF-1,while the underlying connective tissue was negative.
Sixteen complete medical records were obtained. Thirteen patients underwent preoperative embolization: 1 patient, 3 weeks before surgery; the remainder, 1 to 10 days before surgery. Three patients did not undergo preoperative embolization. The medical records of 3 patients were incomplete. Based on the year the surgery was performed and the prevailing practice of the surgeon at that time, these 3 patients most likely did not undergo embolization.
COMMENT
Transforming growth factor 1 is a polypeptide growth factor that is produced in fibroblasts, macrophages, and endothelial cells. Its role in the pathogenesis of neoplasms is complex. There is strong evidence that a complex network–like interaction between cytokines, growth factors, and other mediators is responsible for cell growth and differentiation. Growth factors such as TGF-1 probably play important immunoregulatory roles. For example, TGF- may stimulate interleukin 6 production in peripheral blood mononuclear cells and keratinocytes.8
Several lines of evidence suggest that alterations in TGF-1 expression may play a key role in the development of JNA. Transforming growth factor 1 exerts control on the cell cycle from the G1 phase to the S phase. This process is mediated by a rapid reduction of the c-myc proto-oncogene and by inhibition of the G1 cyclin/cyclin-dependent kinases by TGF-1.9 There is a synergistic effect between the loss of TGF-1 responsiveness and mutations caused by initiation with a carcinogen, leading to endogenous tumor promotion in initiated cells only.9
Transforming growth factor 1 is also known to induce increased expression of itself. This positive feedback state must be countered in the normal physiologic state. Thus, it is not difficult to imagine a situation in which alterations in TGF-1 expression would lead to accelerated cell growth. Tumor inhibition and promotion involve a complex relationship between TGF-1 and TGF-1 receptor. Other cytokines and growth factors are also implicated in the regulation of TGF-1. Any relative deficiency of a receptor for TGF- or any imbalance in the relationship of one cytokine to another can dramatically alter the response of a tissue to a given concentration of TGF- in the tissues. For example, expression of a dominant negative type II TGF- receptor in mouse skin results in an increase in carcinoma incidence and an acceleration of carcinoma development.9 An example of the differential response of tissues to TGF-1 is the proposed alteration in scar formation related to alterations in the ratio of TGF-1 to TGF-3.10 The antiproliferative effect and the migration-stimulatory activity of TGF-1 and prostaglandin E2 suggest a role in the morphogenic processes of angiogenesis.9
In summary, TGF- helps regulate the cell cycle, produces extracellular matrix deposition, and induces angiogenesis, while increasing its own expression. Thus, the identification of TGF-1 in JNA provides ample avenues for future research. Other substances, including the proteoglycan decorin, can block the activity of TGF- in tissues.11 Therefore, if TGF- can be implicated in the pathogenesis of JNA, then the potential for arresting the growth of extensive tumors may permit safer extirpation of the more extensive tumors. Our study identified the immunolocalization of activated TGF-1 in significant percentages of JNA stromal cells. We conclude that TGF-1 may play a significant role in the pathogenesis of JNA.
CONCLUSIONS
Juvenile nasopharyngeal angiofibroma, which is an uncommon tumor that affects male adolescents, results in significant morbidity and mortality. Activated TGF-1 has been identified within the stromal and endothelial cells of JNA, which suggests a possible role for TGF-1 in the pathogenesis of JNA. Investigating the role of TGF-1 may provide more effective methods of managing extensive JNA tumors.
AUTHOR INFORMATION
Accepted for publication December 12, 1999.
This study was supported in part by the University Research Fund, Emory University School of Medicine, Atlanta, Ga.
Corresponding Author: Anthony A. Gal, MD, Department of Pathology and Laboratory Medicine, Emory University School of Medicine, 1365 Clifton Rd NE, Atlanta, GA 30322.
From the Departments of Otolaryngology–Head and Neck Surgery (Drs Dillard, Muller, DelGaudio, Reichman, and Parrish and Mr Rackley) and Pathology and Laboratory Medicine (Drs Cohen, Muller, and Gal), Emory University School of Medicine, Atlanta, Ga.
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