Fibrodysplasia ossificans progressiva

* Background.-Juvenile nasopharyngeal angiofibroma is a rare nasopharyngeal tumor that occurs exclusively in adolescent boys. It is a histologically benign but locally persistent growth of stromal and vascular tissue. Although male hormones and some growth factors, such as transforming growth factor [beta]1 (TGF-[beta]1), insulin-like growth factor II (IGF-II), and, lately, the proto-oncogene [beta]-catenin, have been implicated in the histogenesis of the tumor, the biologic signaling pathways that drive this peculiar fibrovascular proliferation are still nuclear.

Objective.-To evaluate immunoexpressions of [beta]-catenin, c-Kit, p130Cas, TGF-[beta]3, bone morphogenic protein 4, nerve growth factor (NGF), and the IGF receptor (IGF-1R) in a series of juvenile nasopharyngeal angiofibromas and to compare to that of a group of nasal polyps.

Design.-A standard immunohistochemical technique was used on paraffin sections of 12 sporadic juvenile nasopharyngeal angiofibromas and 15 nasal polyps with microwave or steam antigen retrieval. Immunoreactivity was analyzed semiquantitatively in stromal cells and endothelial cells of each case.

Results.-The expressions of [beta]-catenin (nuclear), c-Kit (cytoplasmic), and NGF (cytoplasmic) were higher and more frequent in stromal cells of juvenile nasopharyngeal angiofibromas than those of nasal polyps. Both juvenile nasopharyngeal angiofibromas and nasal polyps showed similarly frequent and strong immunoreactivity for p130Cas andTGF-[beta]3 and weak immunoreactivity for bone morphogenic protein 4 in both stromal cells and endothelial cells. No IGF-1R immunoreactivity was detected in any case of either group.

Conclusions.-Our results support the role of [beta]-catenin in juvenile nasopharyngeal angiofibromas and suggest a potential involvement of c-Kit and NGF signaling pathways in the juvenile nasopharyngeal angiofibromas. Although the biologic significance of c-Kit in juvenile nasopharyngeal angiofibromas has yet to be defined, the finding of frequent and high c-Kit expression might have therapeutic importance for patients with juvenile nasopharyngeal angiofibromas.

Juvenile nasopharyngeal angiofibroma (JNA) is a rare nasopharyngeal fibrovascular tumor that occurs exclusively in the nasopharynx of adolescent boys.1-3 Histologically, it is composed of poorly defined but bland stromal cells and an irregular vascular proliferation.4 Despite its bland histologic features, JNA typically behaves as a locally aggressive tumor. JNA is currently treated surgically, but local recurrence is common and has been reported in up to 42% of cases.3,5,6

The histogenesis of JNA is still unclear; however, because of the exclusive male predilection, sex hormones have been implicated.7,8 Androgen receptor has been detected in the stromal cells of JNA.9-11 Antiandrogen hormonal therapy has been used preoperatively to suppress JNA growth and enhance the resectability of the tumor and minimize bleeding during surgery.1,12,13

P130Cas, an adapter and docking protein, has been shown to be involved in intracellular signaling pathways related to cell adhesion, migration, and transformation.14,15 Recently, high p130Cas levels have been associated with resistance to first-line tamoxifen treatment in patients with advanced estrogen-dependent breast cancer.16,17 Its expression has not been evaluated in JNA, which commonly expresses androgen receptor.

Familial adenomatous polyposis (FAP) results from germline mutations in the adenomatous polyposis coli (APC) gene that subsequently alters the [beta]-catenin signaling pathway.18 In addition to a high tendency for the development of colorectal neoplasms, patients with FAP develop JNA 25 times more frequently than an age-matched population.19,20 A role for the APC-[beta]-catenin pathway has been suggested in JNA patients with the APC gene mutation. Recently, activating [beta]-catenin mutation without the APC gene mutation has been reported in sporadic JNA.21,22

Expression of various growth factors, such as transforming growth factor [beta]1 (TGF-[beta]1) and insulin-like growth factor II (IGF-II), has also been detected in JNA.23,24 IGF functions via the receptor IGF-1R, which has been implicated in tumorigenesis, at least partly as a result of promoting cell survival in both tissue culture and animal study.25 However, it is unclear whether IGF-1R is expressed in JNA. Bone morphogenic proteins (BMPs) are members of the TGF-[beta] superfamily and have a complex role in regulating cell growth and differentiation via the BMP-Smad signaling pathway.26 BMP4 has been shown to regulate steroidogenesis in animal and human gonads.27 Overexpression of BMP4 has been found in patients with fibrodysplasia ossificans progressiva and in various bone and soft tissue sarcomas.26,28 Little is known about the potential role of BMP in the stromal proliferation in JNA.

Proto-oncoproteins c-Kit (CD117) is tyrosine kinase receptor that belongs to the family of platelet-derived growth factor receptors (PDGFRs).29 Expression of c-Kit and activation mutation of the gene have been detected in some mesenchymal tumors, such as gastrointestinal stromal tumors.30,31 Recently, a tyrosine kinase inhibitor specific for the PDGFR family (STI571) has been reported to have therapeutic effects in tumors that express either aberrant forms or high quantities of the corresponding target protein.31,32 So far, c-Kit expression has not been evaluated in JNA.

In addition to its well-known effect to neural tissue, nerve growth factor (NGF), a member of the neurotrophin family, has been shown to have regulatory effects on mast cell differentiation and angiogenesis in inflammatory conditions through its receptor TrkA.33,34 Fibroblasts of the human lower respiratory tract have been shown to secret NGF.35 The NGF-Trk signaling has also been speculated to play a role in tumorigenesis in some solid tumors.36 The role of NGF-TrkA signaling has not been evaluated in JNA that is composed of prominent fibroblastic stromal cells and vascular proliferation of the upper respiratory tract origin.

Nasal polyps are a benign inflammatory and reactive stromal growth that involve nasal cavity and paranasal sinuses and are usually associated with underlying allergic conditions or chronic inflammation.37 Recurrence is common after surgery, which is sometimes used to relieve the symptoms. However, in contrast to JNA, nasal polyps are usually nondestructive and self-limited after elimination of the underlying allergens or the cause of the chronic inflammation.38

To investigate the possible role of these factors in the pathogenesis of JNA, we examined expression of these factors in a series of JNAs by immunohistochemical methods. For comparison, a group of benign nasal polyps was also examined. We compared immunoreactivity of these factors in both the stromal cells and endothelial cells between JNA and nasal polyps. In this article, we report our results and indicate the importance of some of these factors and the absence of significant expression of others.

MATERIALS AND METHODS

Records of 12 cases of JNA and 15 cases of nasal polyps were retrieved from the surgical pathology files at the University of Pennsylvania Medical Center, Philadelphia. All patients with JNA were male and ranged in age from 11 to 23 years old (average age, 17 years) at the time of the surgery. None of the patients had a history of FAP. Of the 15 patients with nasal polyps, the malefemale ratio was 2:3, and patients ranged in age from 21 to 87 years (average age, 58 years). All the patients with nasal polyps had acute and chronic inflammatory conditions in the nasal and/ or sinonasal cavity and 3 (20%) had recurrences after initial surgery. A representative paraffin block was selected from each case on histologie review of the hematoxylin-eosin-stained slides. Four-micrometer-thick sections were cut onto probe-on slides (Fisher Scientific, Pittsburgh, Pa) for immunohistochemical staining. Immunodetection of [beta]-catenin, c-Kit, p130Cas, TGF-[beta]3, BMP4, NGF, and IGF-1R was performed using either monoclonal or polyclonal antibodies with a microwave or steam antigen retrieval method as summarized in Table 1. Primary antibodies against [beta]-catenin, c-Kit, p130Cas, and IGF-1R were incubated at room temperature for 30 minutes, and the immunostaining was completed on a Dako autostainer with Envision Plus software (Dako Corporation, Carpinteria, Calif). Antibodies against TGF-[beta]3, NGF, and BMP4 were incubated at 4[degrees]C overnight, and the SAHRP method was used manually for immunodetection. The intensity of the immunoreactivity and the percentage of positive cells were initially estimated semiquantitatively under light microscope in stromal cells and endothelial cells of JNA and nasal polyps in a semiquantitative method as previous described.39,40 The overall immunoreactivity was then scored in a 4-tier scale system based on the percentage of positive cells and the intensity of immunoreactivity. Immunoreactivity was scored as follows: -, negative result; +, weak immunoreactivity regardless of the percentage of the cells being positive; ++ , moderate immunoreactivity in less than 75% or strong immunoreactivity in less than 25% of the cells; and +++, moderate immunoreactivity in 75% or more or strong immunoreactivity in 25% or more of the cells. Only cytoplasmic reactivity was considered specific for c-Kit, p130Cas, TGF-[beta]3, NGF, BMP4, and IGF-1R. Either cytoplasmic or nuclear reactivity was considered specific for [beta]-catenin. The final overall scores for the immunoreactivity of each biologic marker were compared between JNA and nasal polyps, and statistical analysis was performed with the 2-tailed Mann-Whitney U test for the nonparametric data using GraphPad Prism Version 3.0 software (GraphPad Software Inc, San Diego, Calif).

RESULTS

Results of the immunohistochemical analysis are summarized in Table 2. When present, [beta]-catenin immunoreactivity was always cytoplasmic and nuclear in the stromal cells of JNA but only cytoplasmic in stromal cells of nasal polyps and endothelial cells of both JNA and nasal polyps (Figure 1). Immunoreactivity of c-Kit was identified in the stromal cells only, whereas immunoreactivity of BMP4, NGF, p130Cas, and TGF-[beta]3 was seen in both endothelial and stromal cell components of both JNA and nasal polyps. No IGF-1R immunoreactivity was identified in any of the JNA and nasal polyp cases.

Except nuclear [beta]-catenin expression, none of the markers tested were exclusively positive in JNA but not in nasal polyps. In general, differences in immunoreactivity for these biologic markers between JNA and nasal polyps were mainly observed in stromal cells. There was a significant difference in expression of [beta]-catenin (P .10). When stromal cells were compared with endothelial cells in the same group, a statistically significant difference was detected in immunoreactivity of p130Cas in nasal polyps (P = .007).

COMMENT

In this study, we evaluated the potential roles of [beta]-catenin, c-Kit, p130cas, TGF-[beta]3, NGF, IGF-1R, and BMP4 in the growth of JNA by comparing the immunoreactivity of these biologic markers in JNA to that in nasal polyps. Of the markers tested, [beta]-catenin (+++, 92%), c-Kit (+++, 75%; ++, 25%), p130Cas (+++, 42%; ++, 50%), and TGF-[beta]3 (+++, 92%; +, 8%) were strongly expressed, and BMP4 (++, 25%; +, 67%) and NGF (++, 67%; +, 25%) were moderately expressed in the stromal cells of JNA. However, only the [beta]-catenin, c-Kit, and NGF expressions were significantly higher than that of the nasal polyps, a benign inflammatory growth. Strong [beta]-catenin nuclear expression was frequently detected in sporadic JNA but not in any of the nasal polyps. These findings support the role of the [beta]-catenin pathway in JNA via activating the [beta]-catenin gene mutation independent of the APC gene mutation as described previously.21

In addition, a higher frequency of stronger c-Kit expression was identified in JNA than nasal polyps. High levels of c-Kit expression due to activating gene mutation have been found in other solid tumors, such as the gastrointestinal stromal tumor, and have been used as a target for treatment with STI571.31-33 Little is known about the role of the PDGFR-c-Kit pathway in JNA. Our finding of strong c-Kit expression in JNA, however, suggests a possible involvement of c-Kit in JNA, which can be the potential target for treatment with STI571. Traditionally, JNA has been treated mainly with surgery, with a high incidence of local recurrence. Antiandrogen therapy has been added preoperatively to enhance the resectability of the tumor and reduce the local recurrence; in fact, antiandrogen therapy itself has been shown to reduce the size of the tumor without surgery.10 The finding of frequent strong expression of c-Kit in JNA suggests a potential application of STI571 in JNA as the treatment of choice in addition to surgery and antiandrogen therapy to improve the local control of the disease.

Previous studies have shown that fibroblasts in the lower respiratory tract were capable of secreting NGF.36 In this study, we detected NGF immunoreactivity in the fibroblast stromal cells and the endothelial cells in both JNA and nasal polyps, suggesting that the fibroblasts in the upper respiratory tract might also be able to secret NGF in both inflammatory and neoplastic conditions. However, stromal NGF immunoreactivity was significantly stronger in JNA than nasal polyps. NGF has been shown to be able to induce angiogenesis.34,35 This finding indicates a potential role of increased NGF secreted by the stromal cells in promoting vascular growth in JNA.

Although expression of p130Cas and BMP4 was either strong or moderate in JNA, the lack of difference in expression of these growth factors between JNA and nasal polyps suggests an insignificant role of these factors in JNA. Although TGF-[beta]1 and IGF-II were detected in JNA previously,23 the lack of differential expression of TGF-[beta]3 between JNA and nasal polyps and lack of IGF-1R in JNA in our study imply a limited role of these growth factors in JNA.

Although morphologic analysis of JNA shows that the stromal cells are bland and the blood vessels appear abnormal, our results indicate that the stromal cells are more likely the elements involved in the histogenesis of the tumor as previously suggested.21 By using comparative immunohistochemical methods, nuclear expression of [beta]-catenin was only detected in the stromal cells of JNA, which also expressed a significantly higher level of c-Kit and NGF than that of their counterparts in nasal polyps. These findings support the notion that the stromal cells rather than the vascular component are neoplastic and responsible for the growth of JNA.

In conclusion, we have first shown a significantly high level of c-Kit immunoexpression in JNA. The role of c-Kit should be further evaluated with molecular methods, and its strong expression in JNA might be potentially important in offering STI571 as an alternative treatment for patients with JNA. The strong [beta]-catenin expression found in our study is in agreement with the results of a previous study and further supports the role of [beta]-catenin in JNA.21 Because the nuclear expression of [beta]-catenin was only present in the stromal cells of JNA but not the nasal polyps, detection of nuclear [beta]-catenin might be helpful in diagnosing small volumes of diseased tissue in the background of reactive or inflammatory stromal cell proliferation as commonly seen in cases with residual or recurrent disease. It is possible that the NGF-TrkA signaling is also involved in JNA because of its strong immunoreactivity in the stromal cells of JNA. Stromal cells are likely the neoplastic component of the JNA that is probably driven by multiple signaling pathways, except those involving p130Cas, BMP4, TGF-[beta]3, and IGF-1R.

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Paul J. Zhang, MD; Randal Weber, MD; Ho-Hi Liang, MD; Teresa L. Pasha, BS; Virginia A. LiVolsi, MD

Accepted for publication June 25, 2003.

From the Departments of Pathology and Laboratory Medicine (Drs Zhang, Liang, and LiVolsi and Ms Pasha) and Otorhinolaryngology (Dr Weber), University of Pennsylvania Medical Center, Philadelphia.

Reprints: Paul J. Zhang, MD, Department of Pathology and Laboratory Medicine, 6 Founders, Hospital of University of Pennsylvania, Philadelphia, PA 19104 (e-mail: pjz@mail.med.upenn.edu).

Copyright College of American Pathologists Nov 2003
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