Tumors displaying hybrid schwannoma and neurofibroma features in patients with neurofibromatosis type 2

Although schwannoma and neurofibroma tumors are generally reported as distinct pathologic diagnoses, sporadic schwannoma/neurofibroma hybrid nerve sheath tumors have been reported in the general population with components of both entities. We report the clinicopathological features of these hybrid nerve sheath tumors in patients with neurofibromatosis type 2 (NF2). A retrospective review of nerve sheath tumor surgical specimens from patients with NF2 enrolled at the National Institutes of Health was performed. Those specimens reported to have schwannoma-like and neurofibroma-like features were selected for further characterization by morphology, immunohistochemical panel (CD34, S100, neurofilament triplet protein (immunostain) (NFTP), epithelial membrane antigen (EMA)), and confirmation as hybrid tumors. Of 43 total NF2 patients undergoing resection of nerve sheath tumors, 11 specimens from 11 (26%) patients were found to be benign nerve sheath tumors exhibiting hybrid features of both neurofibroma and schwannoma. Immunohistochemical studies showed the schwannoma component to be S100+, CD 34- while the neurofibroma component was CD34+, variable S100+. Our experience emphasizes the importance of including this distinct tumor subtype, the schwannoma/neurofibroma hybrid tumor, in the differential diagnosis of nerve sheath tumors in NF2 patients and suggests that the relationship between neurofibroma and schwannoma tumors is closer than previously suspected.


Introduction
Neurofibromatosis type 2 (NF2) is an autosomal dominant disease that is caused by loss-of-function mutations in the NF2 gene located on chromosome 22q12.2. NF2 occurs in ~ 1 in 40,000 people [1]. The NF2 mutation limits the function of the tumorsuppressor protein, merlin (schwannomin), which regulates proliferation of many cell types of neural crest lineage.
Patients with NF2 are at increased risk of developing schwannomas, meningiomas, and gliomas, such as astrocytomas [2]. Schwannomas are the most common type of tumor seen in NF2. Neurofibromas have rarely been described in the literature in association with NF2 and are more commonly seen in neurofibromatosis type 1 (NF1) [3]. Unlike schwannomas, these tumors arise within the neural fascicles.
Although sporadic hybrid schwannoma/ neurofibroma tumors have been described, schwannoma/neurofibroma hybrid tumors in the setting of NF2 have rarely been reported in the literature, and their pathology has not been fully described [4,5,6,7,8,9]. We report a series of patients with NF2 harboring schwannoma-neurofibroma hybrid nerve sheath tumors.

Materials and methods
A retrospective review of all resected nerve sheath tumor specimens from patients with NF2 enrolled in the Natural History Protocol #08-N-0044 at the Clinical Center, National Institutes of Health between 2007 and 2013 was performed. Pathologists (MQ, MM, MA) selected formalin fixed paraffinembedded (FFPE) tissue of surgical specimens that, on original histological review, had features of both schwannoma and neurofibroma. The FFPE tissue was sectioned and immunohistochemical staining was performed using a panel of antibodies consisting of CD34, S100, neurofilament triplet protein (immunostain) (NFTP), and epithelial membrane antigen (EMA). Immunohistochemical procedures were performed using an automated immunostainer following the manufacturer's specifications. Specimens displaying immunohistochemical features of both schwannoma and neurofibroma were then confirmed as schwannoma/neurofibroma hybrid tumors. Further analyses of patients with hybrid tumors were then performed, including recording patient demographics, tumor location, clinical and radiographic features, and long-term follow-up. Clinical notes, intraoperative reports, and imaging of the selected patients were analyzed.

Results
43 patients with NF2 underwent surgery for removal of one or more nerve sheath tumors. 28 patients had surgery for non-vestibular intracranial schwannomas or peripheral nerve sheath tumors, 10 patients had surgery for vestibular schwannomas, and 5 patients had surgery for both vestibular and non-vestibular schwannomas. On microscopic evaluation, 11 specimens from 11 (26%) patients had benign nerve sheath tumors exhibiting hybrid features of both neurofibroma and schwannoma. The 11 patients had a median age of 35 years (range 12 -52 years) and included 7 females and 4 males. The tumors were most commonly located in the extremi-   Wavy nuclei, focal nuclear atypia Immunophenotype S100+; CD34-CD34+; S100 +/-(variable); NFTP stains residual axonal processes Additional features Hyalinized vessels with occasional thrombosis; degenerative areas may be histiocyte-rich with hemosiderin representing ischemic infarction Collagenous background stroma may be myxoid Figure 2B.  ties, with some involving the head, neck, and trunk, with size varying from 0.3 cm to 8.4 cm in diameter. These clinicopathological features have been summarized in Table 1.
Eight of these patients presented with pain while 3 developed neurologic symptoms related to tumor growth. Eight patients had multiple contiguous tumor nodules noted on imaging. Gross total resection was achieved in 10 cases and no intraoperative complications occurred. Patients received post-operative clinical and radiographic follow-up for an average of 36 months (6 -118 months). No gross signs of recurrence or delayed complications occurred during follow-up. Symptomatic relief was noted in all cases.

Imaging findings
T1-weighted magnetic resonance images with contrast were reviewed for all patients. All of the tumors were enhancing (Figure 1). Measurements are listed in Table 1.

Microscopic findings
Pathology review of these 11 specimens showed dual histologic components of both schwannoma and neurofibroma ( Table 2). Schwannoma histology, incorporating highly ordered cellular components (Antoni A) with loose, myxoid components (Antoni B) were the distinctive features with a minor component demonstrating neurofibromalike features [10]. Areas of neurofibroma had wavy nuclei interspersed with collagen fibers. Degenerative changes including focal cellular atypia, hyalinization of blood vessels and aggregation of histiocytes were variably present. Tumors were further characterized by immunohistochemical studies with S100, CD34, NFTP, and EMA. Scattered cellular atypia was present in a few sections but mitotic activity was very low and necrosis absent. The observed dual histology of these hybrid tumors was further supported by a corresponding dual immunophenotype: the schwannoma component was S100 positive and CD34 negative while the neurofibroma component contained CD34 positive fibroblasts and S100 protein-positive Schwann cells (Figure 2A, B). Immunostain for NFTP highlighted residual axonal processes in the neurofibroma component. EMA was negative in both components, showing no evidence for a perineurioma in these hybrid tumors. However, an EMA-positive perineurioma was removed from the finger of 1 patient who also had a separate hybrid schwannoma/neurofibroma.

Discussion
Schwannomas were first described in the early 1900s by Verocay and Antoni [11,12]. NF2 schwannomas are most commonly located on the vestibular nerve (bilateral vestibular schwannomas), but can also manifest on other cranial nerves, spinal nerves, and peripheral nerves [13,14,15,16]. On gross examination these tumors are encapsulated by epineurium and on cut section can vary in color from pink to yellow white [10]. Histologically, schwannomas contain two distinct components. Antoni A areas consist of compact cellular regions consisting of nuclear palisading, cell whorling, and Verocay bodies (areas with rows of nuclei separated by fibrillary cell processes) while Antoni B areas represent hypocellular areas with less order and a more loosely structured matrix [10,17,18]. Degenerative changes may be seen in Antoni B areas with hemosiderinladen histiocytes and collagenous fibrosis [17]. Immunohistochemical studies show that schwannomas stain positive for S100 in both the nucleus and the cytoplasm [17,19]. S100 stains both the Antoni A and B portions of the tumor, with more intense staining of the Antoni A regions [19]. The tumor itself is typically negative for EMA, but EMA positive perineurial cells are common within the capsule [17]. CD34 positivity may be seen in the periphery of the tumor.
Neurofibromas arise from cutaneous and deep nerves, autonomic nerves, and spinal nerve roots and typically exhibit one of three growth patterns: localized, diffuse, or plexiform [10]. Neurofibromas consist of Schwann cells, fibroblasts, histiocytes, and mast cells and may also contain perineurial cells [20]. The tumors commonly incorporate axons throughout a nerve, making complete surgical tumor resection without nerve sacrifice impossible [21,22]. Histologically neurofibromas have smaller wavy nuclei, ~ 1/3 -1/2 size of the nuclei seen in schwannomas. Immunohistochemical studies show neurofibromas to have CD34 positivity, variable S100 positivity, and rare EMA positivity if perineurial cells are present [23].
In a smaller study, hybrid schwannoma/ neurofibroma peripheral nerve sheath tumor was found in 10 of 14 (71%) patients with schwannomatosis [7]. Excluding patients with this diagnosis revealed that 26% of the remaining hybrid tumors occurred in patients with NF2. In our study, there was a 26% incidence of schwannoma/neurofibroma hybrid tumors in patients diagnosed with NF2 that underwent surgical resection. These socalled hybrid tumors represent a rare, but distinct entity affecting the NF2 patient population. Hybrid tumors were located in the peripheral nervous system in all 11 patients. The majority of patients had multiple contiguous tumor nodules on imaging. Tumor size varied greatly among patients. Schwannoma histology predominated, with neurofibroma histology comprising a minor component of the hybrid tumors. The observed dual histology of these hybrid tumors was further supported by a corresponding dual immunophenotype: schwannoma component stained S100 positive and CD 34 negative while neurofibroma component stained CD34 positive with variable S100 expression.
Our findings indicate that hybrid tumors having dual morphologic and immunophenotypic features of schwannoma/neurofibroma may be more prevalent in the NF2 population than previously realized. Increased awareness of this entity will lead to better estimates of its true prevalence. A needle biopsy sample containing histologic features of schwannoma or neurofibroma alone does not exclude a diagnosis of hybrid tumor, because such a small biopsy could sample a single tumor type and miss the other component of a hybrid tumor.

Conclusions
Our experience suggests that the derivation of schwannomas and neurofibromas may be less distinct than classically appreciated. Although Schwann cells are the suspected tumor cell of origin in both cases, the finding of a hybrid pathology supports the notion that these entities may indeed rather represent a spectrum of Schwann cell pathology. The schwannoma/neurofibroma hybrid tumor should be included in the differential diagnosis of nerve sheath tumors in patients with NF2. Surgical resection of these tumors can be performed safely and effectively with low rates of recurrence. Future study of the genetics of this tumor subtype is warranted.

Acknowledgment
This research was supported by the Intramural Research Program of the National Institute of Neurological Disorders and Stroke at the National Institutes of Health (NIH) and through the NIH Medical Research Scholars Program, a public-private partnership supported jointly by the NIH and generous contributions to the Foundation for the NIH from Pfizer Inc., The Doris Duke Charitable Foundation, The Alexandria Real Estate Equities, Inc. and Mr. and Mrs. Joel S. Marcus, and the Howard Hughes Medical Institute, as well as other private donors.